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Extended angiotensin converting enzyme inhibition changes the innervation of renal glomerular afferent arterioles
Journal of the Autonomic Nervous System 77 Ž1999. 114–124 www.elsevier.comrlocaterjans
Extended angiotensin converting enzyme inhibition changes the innervation of renal glomerular afferent arterioles Susan E. Luff ) , Simone B. Young, Warwick P. Anderson Department of Physiology, Monash UniÕersity, Wellington Road, Clayton, Victoria 3168, Australia Received 10 November 1998; received in revised form 21 May 1999; accepted 17 June 1999
Abstract Chronic inhibition of the angiotensin I converting enzyme ŽACE. with enalapril, results in a phenotypic change of the medial cells of renal afferent arterioles from contractile smooth muscle cells to renin containing epithelioid cells. In normal animals, the density of the innervation of the juxtaglomerular renin containing epithelioid cells is much lower compared to the contractile cells. The effector tissues are known to play an important role in determining the pattern and density of their innervation. In this study, we tested the hypothesis that the density of the innervation of the afferent arteriole smooth muscle cells decreases when they change their phenotype from contractile to renin containing epithelioid cells. The results show that the density of the innervation had significantly increased and the association of the terminals with the smooth muscle cells had changed. There were significantly more varicosities around renal afferent arterioles from rabbits treated with enalapril Ž10 mgrkgrh. for 6 weeks Žmean " SEM s 634 " 175 = 10 3rmm2 vessel surface, cf. 329 " 69 = 10 3rmm2 vessel surface in untreated rabbits, P s 0.05., with the number of neuroeffector junctions remaining the same Ž124 " 14 and 164 " 32 = 10 3rmm2 vessel surface. and significantly more non-contacting varicosities Ži.e. lying) 100 nm from the medial cells. Ž74 " 5% and 25 " 7%, respectively; P s 0.003.. Thus, there was no reduction in the innervation of afferent arterioles in which the smooth muscle cells had changed phenotype in response to enalapril treatment as hypothesised. Instead, it would appear that proliferation of the innervation had occurred, with the formation of additional varicosities but these varicosities failed to form neuromuscular junctions. This study has identified a form of neural plasticity in the kidney that has not previously been described. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Kidney; Blood vessels; Nerve growth; Plasticity
1. Introduction The density and form of the sympathetic innervation differs in different tissues. In arteries, the density of neuromuscular junctions Ži.e. ‘innervation density’, see Luff and McLachlan, 1989. is inversely proportional to the vessel diameter. In addition, the proportion of varicosities around vessels that form junctions is very high Ž70–92%. in the small arteriolar vessels ŽLuff et al., 1991. but is much lower in the larger muscular arteries Že.g. - 50% in rat tail artery, Luff et al., 1995. and in the larger elastic arteries, only non-contacting varicosities lying at variable distances
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from the smooth muscle cells have been reported ŽLuff and McLachlan, 1989.. What determines the innervation of different tissues is not understood although there is evidence that the tissue itself plays a major role ŽTodd, 1986.. Similarly, our knowledge of the plasticity of varicosities and neuroeffector connections of peripheral autonomic terminals on their targets of mature animals is limited. However, in vivo fluorescence studies of autonomic ganglia have demonstrated considerable changes occur in both the form of the dendritic arbours and the location of synaptic endings on the surface of identified autonomic ganglion cells when monitored over a period of several weeks ŽPurves et al., 1987.. The media of afferent arterioles from the outer cortical region of the rabbit kidney consists of a single layer of smooth muscle cells. Under normal circumstances, most of
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the cells along the length of the afferent arterioles are contractile smooth muscle cells but typically, they become epithelioid in nature at their distal end and contain large homogeneous electron dense granules of renin. In these cells, contractile elements with filament attachment sites and caveoli, typical of contractile smooth muscle cells, are much reduced or absent ŽTaugner and Hackenthal, 1989.. However, the number of renin containing epithelioid cells in the afferent arterioles may increase markedly during long term stimulation of the renin–angiotensin system, such as with angiotensin I converting enzyme ŽACE. inhibitors or low salt diets ŽIce et al., 1988; Barrett et al., 1989.. The increase in the number of renin secreting cells within the media of preglomerular arterioles results from the conversion of existing contractile smooth muscle cells to the renin containing epithelioid phenotype rather than cell division of the existing renin secreting cells ŽCantin et al., 1977.. In this study, we tested the hypothesis that the change in phenotype of the medial cells of afferent arterioles in response to enalapril treatment affects the innervation of this important renal vessel. The renal afferent arterioles receive a dense innervation predominantly by sympathetic postganglionic axons ŽBarajas and Muller, 1973; Gorgas, 1978; Reinecke and ¨ Forssmann, 1988; Barajas et al., 1992. and these nerves play an important role in regulating glomerular blood flow and hence, filtration rate as well as renin secretion ŽDiBona and Kopp, 1997.. Both the contractile smooth muscle and renin containing epithelioid cells, are innervated ŽBarajas and Muller, 1973; Gorgas, 1978. and physio¨ logical studies have shown that different receptor mechanisms operate post-junctionally in neurally mediated responses of contractile smooth muscle cells and renin containing epithelioid cells ŽDiBona and Kopp, 1997.. More recently, we have shown that renal afferent arterioles have a high density of neuromuscular junctions, it being one of the most densely innervated arteries so far studied ŽLuff and McLachlan, 1989; Luff et al., 1991.. In the normal rabbit kidney, neuromuscular junction density on contractile smooth muscle cells of afferent arterioles is up to 10 times greater than on renin secreting cells in the same vessel ŽLuff et al., 1992.. In this study, we have investigated whether the density of the innervation of the preglomerular afferent arterioles is markedly reduced Ži.e. from their normal high density to the lower density of juxtaglomerular epithelioid cells. when the phenotype of the medial cells is changed from contractile smooth muscle cells to renin containing epithelioid cells. We have used ACE inhibition by administering enalapril continuously for 6 weeks to promote the phenotypic change of these cells. Quantitative ultrastructural techniques developed by us ŽLuff et al., 1992., were employed to compare the innervation of afferent arterioles from control untreated rabbits with those of treated rabbits in which the most of the medial cells had changed to the renin secretory phenotype.
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2. Materials and methods 2.1. Tissue preparation Nine male rabbits aged 20–25 weeks were allocated to either enalapril-treated Ž n s 4. or untreated Ž n s 5. groups. In the first group, enalapril was administered subcutaneously via mini-pumps ŽAlza, Palo Alto, CA, USA. implanted under the skin between the scapulae under local anaesthesia ŽLignocaine, Delta West, Western Australia.. Mini-pumps were changed at 4 weeks to ensure a continuous infusion of enalapril ŽMaleate salt. of 10 mgrkgrh for 6 weeks. At 6 weeks, all rabbits were anaesthetized with pentobarbitone Ž60 mgrkg, i.v.. and prepared for perfusion fixation of the kidneys using the method described previously ŽLuff et al., 1987.. Catheters were inserted in the central ear artery and vein to measure arterial blood pressure responses to a bolus intravenous injection of angiotensin I ŽAng I. Ž10 ngrkg for untreated control animals and 100 ngrkg for treated animals. to establish the extent of ACE inhibition in enalapril-treated rabbits compared to untreated control rabbits. In all animals, the blood pressure was allowed to return to pre-injection levels. A bolus injection of 3 ml of 1% sodium nitrite plus 5000 IU of heparin was then given via the ear vein to dilate the vasculature. The kidneys were immediately perfused via the abdominal aorta with a fixative solution containing 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer at pH 7.3 at a pressure of 130 mm Hg for approximately 10 min. The kidneys were removed and tissue from the outer cortical region was cut into small pieces Žapproximately 2 mm wide, 3 mm long and 1 mm thick. and fixed at room temperature for a further 2 h in the same fixative. The tissue was then washed in buffer and postfixed in buffered 2% osmium tetroxide for 4 h, dehydrated and embedded in epoxy resin as described previously ŽLuff and McLachlan, 1988.. 2.2. Tissue sectioning and sampling Thin Ž1 mm thick. sections were prepared from blocks of outer cortical tissue to locate afferent arterioles cut in longitudinal section along their entire length Ži.e. from the origin with the interlobular Žcortical radial. artery to the connection with the glomerulus. ŽFig. 1.. Similarly oriented afferent arterioles were selected for ultra microtomy from untreated and enalapril-treated rabbits, but in addition, enalapril-treated afferent arterioles were selected that had most Žmean " SEMs 93 " 4%. of the medial cells packed with renin secretory granules ŽFig. 1B., which was confirmed in low power electron micrographs Žmagnification s =2000; see Fig. 1B.. These selection criteria maximised the vessel adventitial surface present in each section, reducing the number of sections required for each analysis.
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Fig. 1. Low power electron micrographs of two afferent arterioles cut in longitudinal section. ŽA. An afferent arteriole from a control rabbit with the media largely consisting of contractile smooth muscle cells. ŽB. An afferent arteriole from an enalapril-treated rabbit with the media largely consisting of renin containing epithelioid cells. dt, Distal tubule; g, glomerulus; I, intralobular artery; l, lumen of afferent arteriole; pt, proximal tubule. Calibration bar in B s 20 mm and also applies to A.
Ultra-thin sections Ž; 100 nm thick. were prepared from selected enalapril-treated and untreated afferent arterioles in one of two ways: Ži. short ribbons of ; 10 sections, taken every 2 m Ži.e. four to six ribbons of sections per afferent arteriole. from nine enalapril-treated and nine untreated afferent arterioles, Žii. serial sections cut through 2.3–8.7 mm of tissue Ži.e. 23–87 sections. from the mid region of each of four untreated and three enalapril-treated afferent arterioles obtained from three rabbits in each case. Section thickness was determined from the interference colour of individual sections floating on water ŽPeachey, 1958. recorded at the time of cutting Ži.e. pale gold s approximately 100 nm. and used to calculate the surface area analysed in each section Žsee below.. A previous analysis of rabbit afferent arterioles had shown that the size of varicosities ranged from 0.25 to 1.23 mm Žsee Table 1, Luff et al., 1991. hence, a separation of ) 2
mm between sections in Ži. above was chosen to ensure that the same varicosity was not measured twice. 2.3. Analysis Varicosities and neuromuscularrneuroeffector junctions were defined as follows: Õaricosities are axon profiles G 0.25 mm in diameter that contained small Ž50 nm. synaptic vesicles ŽFigs. 2 and 3.; neuromuscular r neuroeffector junctions are varicosities in contact with either a vascular smooth muscle cell Žneuromuscular. or renin containing epithelioid cell Žneuroeffector., with the intervening gap being - 100 nm and containing a single basal lamina Žsee Luff et al., 1991. ŽFig. 2.. Axons were identified as associated with afferent arterioles if they were bare of their Schwann cell along their surface facing the arteriole and lay - 1 mm from an arteriole. These criteria resulted from previous studies on the innervation of the
S.E. Luff et al.r Journal of the Autonomic NerÕous System 77 (1999) 114–124 Table 1 Vessel diameter, vessel length and vessel surface area per vessel length of afferent arterioles from enalapril-treated and untreated rabbits Treatment
Diameter Žmm.
Vessel length Žmm.
Vessel surface arearmm length of vessel Žmm2 rmm.
Control vessels Enalapril-treated vessels P values
20.8"2.5, ns10 33.0"8.6, ns10
121.6, ns9 101.8, ns9
33.0, ns 4 30.8, ns 4
0.001
0.55
0.63
Measurements of vessel surface arearunit length of vessel were taken from sections that were cut through the mid region of the vessel through the point where they entered the glomerulus and connected with the interlobular artery.
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outer cortical arterioles showing that most Ž) 70%. varicosities formed neuroeffector junctions and lay - 1 mm from the cells that they eventually contacted ŽLuff et al., 1991.. In cases where arterioles lay - 1 mm from a tubule, adjacent sections were used to establish whether the synaptic vesicles were clustered towards the afferent arteriole or the adjacent tubule. 2.3.1. Measurement of the proportion of Õaricosities that formed junctions Serial thin sections were used for these analyses. A total medio-adventitial surface area ranging from 1190 to 2520 mm2 Žmean " SEM s 2134 " 821 mm2 , n s 3. for enalapril-treated and 436–2442 mm2 Žmean " SEM s
Fig. 2. Electron micrographs of two sympathetic neuroeffector junctions. ŽA. On a contractile smooth muscle cell of a control afferent arteriole and ŽB. on a smooth muscle cell that has converted to a renin containing epithelioid cell of an afferent arteriole from an enalapril-treated rabbit. In both cases, the varicosities have extended regions of their membrane apposed to the effector cell membrane with the intervening gap being F 100 nm and containing a single basal lamina. Note that the cytoplasm of the smooth muscle cells in A, typically have dense bands and dense bodies and contractile filaments throughout the cell, whereas the epithelioid cell in B, is packed with large electron dense renin granules, endoplasmic reticulum and ribosomes, the contractile filaments being reduced and restricted to a region just under the cell membrane. rc, Renin containing epithelioid cell; sm, contractile smooth muscle cell; v, varicosity. Calibration bar in B s 1 mm and also applies to A.
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Fig. 3. Electron micrographs of non-contacting varicosities. ŽA. An example of a non-contacting varicosity from an untreated afferent arteriole. It is typically small, lacks a mitochondrion and does not have any obvious clustering of vesicles normally associated with vesicle exocytosis. ŽB. A large non-contacting varicosity from an enalapril-treated afferent arteriole which has a clustering arrangement of synaptic vesicles associated with electron dense thickenings of the varicosity membrane Žarrows. typical of a presynaptic membrane specialisation ŽPMS. associated with vesicle exocytosis. Calibration bar in B s 1 mm and also applies to A. rc, Renin containing cell, rg, renin granule, sm, smooth muscle cell, v, varicosity.
1211 " 509 mm2 , n s 4. for untreated afferent arteriole was analysed. The combined area for all the afferent arterioles analysed from the enalapril-treated and untreated group was 6401 mm2 and 4843 mm2 , respectively. Initially, the adventitia of the afferent arterioles in the mid section of each series of sections was scanned in the electron microscope at 5000 times magnification and all the axon profiles located and photographed. These micrographs were used to locate the axon profiles in each section within the series which were subsequently photographed at a higher Ž=15 000. magnification to produce sets of micrographs for complete varicosities. These sets of micrographs were used to determine the proportion of varicosities around each vessel that formed junctions. 2.3.2. Measurement of Õaricosity Õolume and neuromuscular r neuroeffector contact area Measurements of varicosity volume and junction size were obtained from sets of micrographs obtained from serial sections Žas detailed above. of 14 contacting varicosities randomly selected from three enalapril-treated and three untreated afferent arterioles each from a separate animal, using a graphics tablet and custom computer software ŽLuff et al., 1987.. Similar measurements were made of 24 and 8 non-contacting varicosities around enalapriltreated and untreated afferent arterioles respectively, to compare their size in each group of vessels. 2.3.3. NerÕe density measurements In this study, we will refer to density values for varicosities obtained using single sections, as varicosity density. We have previously defined the density of junctions measured in single sections that are expressed as the
number of junctions per smooth muscle cell surface area analysed, as ‘innervation density’ Žsee Luff and McLachlan, 1989.. In contrast, measurements of the number of junctions per smooth muscle cell surface area analysed in serial sections of tissue containing complete varicosities, is true ‘junction density’. We have consequently retained these definitions. The surface area analysed for each afferent arteriole was obtained by tracing around the outer surface of the smooth muscle cells of each vessel profile in low power Žmagnifications 600 times. electron micrographs using a graphics tablet and custom computer software. The medio-adventitial length obtained was subsequently multiplied by the section thickness to obtain the surface area analysed in each section. The number of varicosities and neuromuscular and neuroeffector junctions was obtained from four to six single sections Ž) 2 mm apart. of nine control untreated and nine enalapril-treated afferent arterioles from four and three rabbits, respectively. The mean surface area of vessels analysed per rabbit were: control rabbitss 189 and 94 mm2 Ž n s 3 vessels. from 2 rabbits respectively, 300 mm2 Ž n s 2 vessels. from a third rabbit and 103 mm2 of a single vessel from a fourth rabbit; enalapril-treated vesselss 149, 126, and 113 mm2 Ž n s 3 vessels from each rabbit.. The individual data obtained from each section was combined for each afferent arteriole and the density values expressed as the number of varicosities or junctions per mm2 of vessel surface area. 2.3.4. Afferent arteriole measurements To determine whether enalapril treatment led to changes in diameter and or surface area of afferent arterioles, the outer vessel dimensions were measured from transverse
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sections of 10 afferent arterioles in tissue from two enalapril-treated and two untreated rabbits. The mean diameter of each afferent arteriole was determined from measurements of their maximum and minimum diameters, and the outer surface of the smooth muscle cells at the medio-adventitial border of the arteriole in each section was measured using a graphics tablet and computer software. Additional measurements were made of vessel length, measured in longitudinal sections from the mid region of afferent arterioles Žfour control and four enalapril-treated. down the centre of the lumen from the point where the vessel enters the glomerulus and its point of branching off the interlobular artery. In the same section, we measured along the outer surface of the smooth muscle cells at the medio-adventitial surface. From these two measurements, we calculated the outer cell surface length per unit vessel length to establish whether the change in cell shape had contributed to the change in medio-adventitial surface area of the vessel. 2.3.5. Statistics The data are presented as means " standard error of the mean. A one-way analysis of variance was used to test the statistical significance between values obtained for enalapril-treated and untreated groups of afferent arterioles. P values F 0.05 were considered to be significantly different. Relations between junction density and afferent arteriole diameter were tested using linear regression analysis. All calculations were performed using Statview ŽAbacus Concepts, Berkeley, CA, USA. and SYSTAT ŽWilkinson, 1990. software.
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These experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. 3. Results The mean arterial pressure was similar for the treated and untreated anaesthetized rabbits Žrange 80–110 mm Hg and 87–94 mm Hg, respectively.. There was no discernible effect on blood pressure following a bolus injection of Ang I of 100 ngrkg in enalapril-treated animals. In contrast, 10 ngrkg Ang I in the untreated control rabbits, increased mean arterial blood pressure by G 15 mm Hg. 3.1. General morphology of outer cortical afferent arterioles Renin secreting cells in the media of arterioles were easily identified in 1 mm thick sections stained with toluidine blue when viewed in the light microscope at =1000 magnification, by the presence of dark blue staining renin granules. In tissue from untreated rabbits, the media of afferent arterioles consisted almost entirely of a single layer of smooth muscle cells of the contractile phenotype with only the one to two cells adjacent to the glomerulus containing renin granules ŽFig. 4.. In tissue from enalapril-treated rabbits, the conversion of contractile smooth muscle cells to the renin containing epithelioid phenotype occurred in the preglomerular rather than the post-glomerular blood vessels, with the afferent arterioles being the ones predominantly affected. However, renin granules were also observed in the medial cells of some
Fig. 4. Electron micrograph of an afferent arteriole from a control rabbit at the region where it connects with the glomerulus showing contractile smooth muscle cells Žsm. and renin containing epithelioid cells Žrs.. g, Glomerulus; l, lumen of afferent arteriole; pt, proximal tubule; dt, distal tubule. Calibration bar s 1 mm.
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interlobular arteries. In the nine afferent arterioles selected for analysis from enalapril-treated rabbits, 93 " 4% of cells contained renin granules Ži.e. 100% of the medial cells in five afferent arterioles, 95% in two, and 85% and 70% in each of two other afferent arterioles.. The cells that did not contain renin granules in these vessels lay closer to the interlobular artery ŽFig. 1B.. 3.2. Vessel dimensions In all the afferent arterioles analysed from enalapriltreated animals, it was notable that they appeared to have greater outside and lumina diameters than those from control untreated animals Žcf. Fig. 1A and B.. Also, the renin secreting cells appeared larger in cross-section compared to the smooth muscle cells of the afferent arterioles from untreated rabbits ŽFig. 1.. The outer diameters of afferent arterioles measured in transverse sections were significantly greater for enalapril-treated compared to untreated control vessels Ž P s 0.001. ŽTable 1.. Similarly, the vessel perimeter measured along the outer surface of the smooth muscle cells in transverse sections was also significantly greater Ž P s 0.003.. The ratio of mean vessel diameter for untreated to enalapril-treated afferent arterioles was 0.64. This value was almost identical to that for the ratio of the vessel perimeter measured in transverse section of afferent arterioles from untreated to enalapriltreated afferent arterioles measured in transverse section Ži.e. 0.63.. Measurements of vessel length Žobtained from longitudinal sections. showed that there was no significant difference between the enalapril-treated and untreated groups of afferent arterioles Žmean s 101.8 and 121.6, respectively, P s 0.55.. Also, there was no significant difference in smooth muscle cell surface length per unit vessel length measured in longitudinal sections of enalapril-treated and untreated groups of vessels. Hence, the increase in vessel surface area in the enalapril-treated afferent arterioles can be predominantly attributed to the increase in vessel diameter. Consequently, it was necessary to take into account these differences in vessel diameter to adjust the axon terminal density measurements obtained for the enalapriltreated group of vessels Žsee below.. 3.3. InnerÕation analysis Junctions were observed on both types of cells, i.e. smooth muscle cells in control afferent arterioles and renin secreting cells in afferent arterioles from enalapril-treated tissue. There was no apparent difference in the structure of the neuroeffector junctions Žcf. Fig. 2A and B.; all contained small synaptic vesicles with some having obvious aggregations towards the region of membrane contact ŽFig. 2B.. Also, a similar proportion of junctions Ž22 " 2% in enalapril-treated and 25 " 6% from untreated afferent arterioles. had presynaptic membrane specialisations ŽFig. 2..
At the regions of contact between the nerve varicosities and either the smooth muscle or renin containing epithelioid cells, the gaps between the cell membranes were - 100 nm wide and contained a single basal lamina. In the afferent arterioles from untreated rabbits, the non-contacting varicosities were small Ž- 1 mm in diameter. ŽFig. 3A. and although they contained synaptic vesicles, none had presynaptic membrane specialisations. Conversely, large Ž) 1 mm in diameter. non-contacting varicosities were observed around the enalapril-treated afferent arterioles and some Ž11%. did have presynaptic membrane specialisations ŽFig. 3B.. Most varicosities contained large granular vesicles. It was noticeable that small Ž50 nm. granular vesicles were present in some individual profiles of varicosities whereas others contained only agranular vesicles Žsee Figs. 2 and 3.. However, further investigation of varicosities in serial sections revealed that although individual profiles only contain agranular vesicles, other profiles of the same varicosity contain granular vesicles. Also, individual axons sometimes had varicosities with only agranular vesicles, and others that contained granular vesicles. We therefore concluded that the presence of granular vesicles was not a suitable criteria for distinguishing nerve types. Data obtained from 6-hydroxydopamine treated tissue from the outer cortex of an enalapril and an untreated rabbit, demonstrated that all the varicosities associated with the afferent arterioles Ž90 varicosities from three different pieces of tissue from each animal. reacted positively, indicating that they were all catecholaminergic. In addition, no negatively reacting varicosities were observed associated with any of the other vessels in the same tissues. 3.3.1. Measurements of Õaricosity size and neuromuscular r neuroeffector contact areas The varicosity volumes and neuroeffector contact areas of contacting varicosities in enalapril-treated and untreated afferent arterioles ranged from 0.12 to 1.79 mm3 and 0.02–1.03 mm3 ; 0.03–1.04 mm2 and 0.02–0.89 mm2 , respectively ŽFig. 5.. These values were not significantly different Ž P s 0.11 and 0.16, respectively.. Conversely, the non-contacting varicosities from enalapril-treated animals were significantly larger than those around the untreated afferent arterioles Žrange s 0.05–2.21 mm3 ; n s 24 and 0.04–0.44 mm3 n s 8, respectively; P s 0.003.. 3.3.2. Density of junctions and Õaricosities The number of varicosities analysed in each afferent arteriole ranged from 19 to 74 Žmean " SEM for enalapril-treated afferent arterioless 44 " 7 and for untreated afferent arterioless 46 " 7.. The total number of varicosities analysed in the enalapril-treated and untreated group of afferent arterioles was similar Ž397 and 413, respectively.. The data obtained for the number of varicosities lying - 1 mm from the surface of afferent arterioles from
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Fig. 5. Histograms of mean values for ŽA. varicosity volume and ŽB. neuromuscular contact area of varicosities innervating afferent arterioles from enalapril-treated and untreated rabbits.
enalapril-treated rabbits, before adjustment for the differences in vessel dimensions Žsee Section 3.2., was 401 " 97 = 10 3rmm 2 of vessel surface and untreated rabbits was 329 " 69 = 10 3rmm2 of vessel surface Ž P s 0.53., with the number of junctions on smooth muscle cells of afferent arterioles from enalapril-treated and untreated rabbits being 84 " 13 and 164 " 32 = 10 3rmm 2 of vessel surface, respectively Ž P s 0.025.. The relationship between the log 10 transformation of ‘innervation density’ measurements Ži.e. the number junctions per mm2 of vessel surface measured using single section analysis. and afferent arteriole diameter was linear Ž r s 0.71; P - 0.0001. ŽFig. 6.. This data together with the data obtained for vessel dimensions Žsee above. demonstrate the necessity to adjust the density measurements for varicosities and neuroeffector junctions in enalapril-treated afferent arterioles for the difference between vessel diameters by multiplying the values for each vessel by the ratio of the mean diameters of afferent arterioles of untreated to enalapril-treated rabbits ŽFig. 7.. The data showed that varicosity density was greater in enalapril-treated rabbits Ž329 " 69 and 634 "
Fig. 6. Linear regressions of the log 10 transformed ‘innervation density’ Žexpressed as number of junctions per mm2 of afferent arteriole vessel surface. and afferent arteriole diameter.
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Fig. 7. Histograms of ŽA. varicosity density and ŽB. innervation density, after adjustment for differences in afferent arteriole diameter. Density values are expressed as number of junctions or varicosities per mm2 of afferent arteriole vessel surface.
175 = 10 3rmm2 of afferent arteriole surface, P s 0.05. whereas the ‘innervation density’ for afferent arterioles from untreated and enalapril-treated rabbits were not significantly different Žmean s 164 " 32 and 124 " 14 = 10 3rmm 2 of afferent arteriole surface, respectively, P s 0.24.. 3.3.3. Measurements of the proportion of total number of Õaricosities that form junctions Measurement of the proportion of varicosities that formed neuroeffector junctions was calculated using serial section analysis of three afferent arterioles Žeach from a different animal. from enalapril-treated rabbits and from four afferent arterioles Žeach from a separate animal. from untreated rabbits. In the afferent arterioles from untreated rabbits, most varicosities Ž75 " 5%. formed junctions with contractile smooth muscle cells of the media. However, the proportion of varicosities that formed junctions with renin secreting cells of the media of afferent arterioles from enalapril-treated rabbits was significantly lower Žmean s 26 " 5%; P s 0.003. ŽFig. 8..
Fig. 8. Histogram of the percentage of varicosities associated with afferent arterioles that form junctions in vessels from enalapril-treated and control untreated animals.
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4. Discussion Extended treatment of rabbits with the angiotensin converting enzyme inhibitor enalapril resulted in changes in the phenotype of many of the vascular smooth muscle cells of renal afferent arterioles Žsee also Barrett et al., 1989., with the media of some vessels consisting entirely of renin containing epithelioid cells. In afferent arterioles selected for study from the outer cortical region of the kidney, ) 90% of the contractile smooth muscle cells had changed their phenotype to renin containing epithelioid cells that contained large electron dense renin granules. The detailed ultrastructural analysis conducted on the innervation of the afferent arterioles in this study encompassed a number of different aspects to ascertain whether there was any quantitative and or structural change to the innervation in the enalapril-treated afferent arterioles compared to control untreated vessels; these included, the structure of the terminals and their relationship with the smooth muscle cells as well as the density of varicosities and neuroeffector junctions around the afferent arterioles. The data clearly demonstrate that the axon terminals innervating afferent arterioles from enalapril-treated rabbits differed structurally, quantitatively and in their relationship with smooth muscle cells from those innervating afferent arterioles from control untreated rabbits. 4.1. Structure of Õaricosities and their association with smooth muscle cells There was a marked difference between the afferent arterioles of enalapril-treated and control animals, in the proportion of varicosities around the vessels that had formed junctions, the proportion being considerably lower Ž26%. for the enalapril-treated compared to the untreated vessels Ž75%.. The proportion of varicosities forming junctions in the control Žuntreated. arterioles was similar to that reported in a previous study Ži.e. 70–93%; see Luff et al., 1991.. This analysis was conducted on serial sections through entire varicosities and consequently, is independent of varicosity size, neuroeffector contact area size and is not related in anyway to the size of the vessel. There was also a difference in the structure of the non-contacting varicosities innervating the afferent arterioles from enalapril-treated rabbits. In the afferent arterioles from untreated rabbits in the current study as well as our previous study ŽLuff et al., 1991., it was notable that the varicosities that did not form junctions were small Ž- 1 mm in diameter., lacked mitochondria and contained only a few synaptic vesicles. Conversely, in the afferent arterioles from enalapril-treated rabbits there were many noncontacting varicosities that were large Ž) 1 mm in diameter. and contained numerous synaptic vesicles, with some having presynaptic membrane specialisations similar to those found around larger muscular arteries ŽLuff et al., 1995.. This suggests that neurotransmitter release may be
occurring at both junctional and extra-junctional sites in the afferent arterioles from enalapril-treated rabbits but is predominantly released at junctions in vessels from control rabbits. 4.2. Density of Õaricosities and neuroeffector junctions The density of varicose terminals around afferent arterioles of enalapril-treated rabbits was significantly greater compared to that around control vessels. In contrast, the ‘innervation density’ values Ži.e. number of neuroeffector junctions per mm2 of vessel surface. were similar for both groups of afferent arterioles. These ‘innervation density’ values were not related to changes in the size of the terminals or the neuroeffector contact areas, as neither of these values differed for the two groups of afferent arterioles. The density values for neuromuscular junctions obtained from analysis of single sections of vessels, is not the true junction density and is greater than the density values obtained from serial sections previously published. In this previous study, we showed that ‘innervation density’ for renal afferent arterioles are approximately eight times greater than junction density values obtained from analyses conducted in serial sections ŽLuff et al., 1992.. Measurements of ‘innervation density’ reflects the frequency of junctions encountered in a single section, whereas measurements in serial sections represents true junction density. The difference in the values is related to the size of the junctions and number of sections in which the junctions fall Žsee Luff et al., 1992.. We previously demonstrated that the density of neuromuscular junctions on afferent arterioles from normal untreated rabbits is much lower Ž; 10%. on the renin secreting cells than on the contractile smooth muscle cells in the same vessel Žmean s 2.25 and 20.0 = 1000rmm2 of cell adventitial surface, respectively ŽLuff et al., 1992... Hence, we predicted that a reduction in the density of junctions would result with the change of cell phenotype resulting from enalapril treatment. Such a result would be in agreement with other studies where the pattern of innervation, including that of blood vessels ŽTodd, 1986. and salivary glands ŽPurves et al., 1988. is regulated by the effector tissue. However, the data indicates that the innervation had proliferated rather than reduced with an increase in the number of non-contacting varicosities but not in the varicosities forming neuroeffector junctions. This is supported by the data obtained from serial sections for the proportion of varicosities forming junctions Žwhich are independent of changes in vessel surface area. showing a significantly greater number of non-contacting varicosities around the enalapril-treated group of afferent arterioles. Hence, it would appear that the phenotype of the cells forming the media of renal afferent arterioles is not important in determining density of axon terminals innervating these vessels at least, after the innervation has been established. Alternatively, autonomic neuromuscular junctions may be stable structures once they have formed. Our knowledge of the
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plasticity of varicosities and neuroeffector connections of peripheral autonomic terminals on their targets of mature animals is limited. Nevertheless, it has been demonstrated in vivo that the distribution of presynaptic endings on the surface of autonomic ganglion cells changes over a period of 1–3 weeks ŽPurves et al., 1987. indicating that autonomic nerves can and do change their synaptic connections at least in ganglia. Further studies on the innervation of afferent arterioles in tissue from enalapril-treated animals that did not exhibit any phenotypic change in their smooth muscle cells would provide information on whether the cell phenotype is important in promoting the sprouting of varicosities and preventing the formation of additional neuroeffector junctions observed in this study. We interpret the results as indicating that varicosities that were originally in contact with contractile smooth muscle cells have remained in contact with them after they have changed phenotype. Furthermore, that new varicosities had developed around the enalapril-treated group of vessels but had not formed junctions with the cells of altered phenotype. This gives rise to the question: why have new varicosities formed? Neurite outgrowth from isolated chick sympathetic neurons has been shown to be inhibited in the presence of Angiotensin II ŽAng II. ŽReed et al., 1996.. Hence, an explanation for the increase in number of varicosities around the enalapril-treated afferent arterioles may lie in the abnormally low level of circulating Ang II resulting from ACE inhibition. The role of Ang II in the growth of nerves in vivo is not fully understood and certainly during development, the opposite case is true in that Ang II promotes nerve growth factor release, which in turn promotes the growth of nerves ŽAlcorn et al., 1996.. However, the receptors involved in this interaction during development are AT2 receptors, which are not normally present in this tissue in adult animals ŽAlcorn et al., 1996.. An explanation for why the newly formed varicosities did not form junctions with smooth muscle cells that have converted to renin containing epithelioid cells could be that the cells with established neuroeffector junctions have rejected axons forming any further neuroeffector contacts. Evidence for this comes from a study of the growth of sympathetic neurons together with smooth muscle cells in culture ŽChamley et al., 1973., which showed that if more than one axon contacted the same smooth muscle cell at approximately the same time, then all axons would form lasting contacts. Conversely, axons that formed contacts with smooth muscle cells that already had established neuromuscular junctions, failed to form lasting contacts. Another possible explanation is that the 6-week treatment period was insufficient for newly formed varicosities around the enalapril-treated afferent arterioles to form neuroeffector junctions, but this is unlikely as during early postnatal life, axon varicosities form and establish neuromuscular junctions on rat mesenteric arteries within 4–8 days ŽLuff, 1999..
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4.3. Adjustment of density measurements for differences in Õessel dimensions The results of this study clearly show that there is a change in the innervation of afferent arterioles in response to enalapril treatment and that this is true whether the data is corrected for the increase in vessel surface area or not. The question being addressed was, ‘‘whether the number of junctions become reduced in response to enalapril treatment in vessels where the smooth muscle cells have changed their phenotype.’’ To quantify this, we have measured ‘innervation density’ which is expressed as the number of junctions per unit area of the outer surface of the smooth muscle cells at the medio-adventitial border. Prior to enalapril treatment, each vessel would have started with a finite number of axon varicosities and neuromuscular junctions. As a result of enalapril treatment, it is evident both from our own studies and others Že.g. Notoya et al., 1996., there is a significant increase in the diameter Žboth internal and external. of afferent arterioles from kidneys that have been subjected to ACE inhibition over a period of several weeks resulting in an increase in the surface area of the vessel at the medio-adventitial border where the nerve terminals are located. Hence, if there is no change in the number of terminals or junctions around each vessel in response to enalapril treatment then because the surface area of the vessels is increased, the density of both junctions and varicosities would be lower than that on the control vessels. This was not the case; the junction density was lower but the varicosity density was unchanged. In order to establish and quantify what had changed and by how much, it was necessary to correct the density measurements for the change in the outer surface area of the smooth muscle cells. We have shown that the outer surface of the smooth muscle cells did change in response to an increase in vessel diameter but did not change due to a change in cell shape or any restructuring of the vessel wall that might have occurred. This was demonstrated by the lack of change in either vessel length or in the cell surface lengthrunit vessel length measured in longitudinal sections through the mid region of the vessel. 4.4. Type of nerÕes The innervation of the cortical vessels is predominantly made up of sympathetic axons ŽGorgas, 1978; Reinecke and Forssmann, 1988; Barajas et al., 1992. but a small population of afferent nerves have also been demonstrated in a number of different animal species. In addition, there is evidence for heterogeneity of the sympathetic component of the innervation both with respect to their peptide content ŽReinecke and Forssmann, 1988; Knight et al., 1989. and structure ŽLuff et al., 1991.. Data obtained from vessels from animals pretreated with 6-hydroxydopamine indicates that the changes to the innervation demonstrated in this study predominantly if not exclusively involve the sympathetic nerves as in both the enalapril-treated and
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untreated tissues, all the axon terminals identified reacted positively with 6-hydroxydopamine Ži.e. contained small granular vesicles.. It is possible that a specific sub population of sympathetic nerves may be involved in the innervation changes demonstrated in this study, but to determine if this was the case, would required extensive investigation which is beyond the scope of this study.
5. Conclusion We have demonstrated a change in the innervation of rabbit afferent arterioles in which the majority of the contractile smooth muscle cells have converted to a renin containing epithelioid phenotype in response to 6 weeks treatment with enalapril. As a result of the enalapril treatment, the innervation of the afferent arterioles in which most of the medial cells were transformed to renin containing epithelioid cells, had altered. In particular, the proportion of total number of varicosities around these vessels that formed neuroeffector junctions was much lower. However, the results of this study did not support our original hypothesis that ‘innervation density’ would be reduced as a result of the change in medial cell phenotype. In fact, the data indicated that there was an increase in the innervation in response to enalapril treatment with more varicosities being formed, but the density of junctions remained the same as that on the untreated vessels. We hypothesise that the newly formed varicosities, failed to form neuroeffector junctions with the renin containing epithelioid medial cells. This study has identified a form of neural plasticity that has not been previously described. The increase in the density of the innervation in response to enalapril treatment is likely to have significant functional effects on renin release which remains to be determined.
Acknowledgements This work was supported by the National Heart Foundation of Australia and the National Health and Medical Research Council of Australia.
References Alcorn, D., McCausland, J.E., Maric, C., 1996. Angiotensin receptors and development: the kidney. Clin. Exp. Pharmacol. Physiol. Suppl. 3, S88–S92. Barajas, L., Muller, J., 1973. The innervation of the juxtaglomerular ¨ apparatus and surrounding tubules: a quantitative analysis by serial section electron microscopy. J. Ultrastruct. Res. 43, 107–132. Barajas, L., Liu, L., Powers, K., 1992. Anatomy of the renal innervation: intrarenal aspects and ganglia of origin. Can. J. Physiol. Pharmacol. 70, 735–749. Barrett, G.L., Morgan, T.O., Smith, M., Alcorn, D., Aldred, P., 1989.
Effect of converting enzyme inhibition on renin synthesis and secretion in mice. Hypertension 14, 385–395. Cantin, M., Araujo-Nascimento, M., Benchimol, S., Desmeaux, Y., 1977. Metaplasia of smooth muscle cells into juxtaglomerular cells in the juxtaglomerular apparatus, arteries and arterioles in the ischemic Žendocrine. kidney. Am. J. Pathol. 87, 581–602. Chamley, J.H., Campbell, G.R., Burnstock, G., 1973. An analysis of the interactions between sympathetic nerve fibres and smooth muscle cells in tissue culture. Dev. Biol. 33, 344–361. DiBona, G.F., Kopp, U.C., 1997. Neural control of renal function. Physiol. Rev. 77, 75–197. Gorgas, K., 1978. Structure and innervation of the juxtaglomerular apparatus of the rat. Adv. Anat. Embryol. Cell Biol. 54, 5–84. Ice, K.S., Geary, K.M., Gomez, R.A., Johns, D.W., Peach, M.J., Carey, R.M., 1988. Cell and molecular studies of renin secretion. J. Clin. Exp. Hypertens. 10, 1169–1187. Knight, D.S., Fabre, R.D., Beal, J.A., 1989. Identification of noradrenergic nerve terminals immunoreactive for neuropeptide Y and vasoactive intestinal peptide in the rat kidney. Am. J. Anat. 184, 190–204. Luff, S.E., 1999. Development of neuromuscular junctions on small mesenteric arteries of the rat. J. Neurocytol. 28, 47–62. Luff, S.E., McLachlan, E.M., 1988. The form of sympathetic postganglionic axons at clustered neuromuscular junctions near branch points of arterioles in the submucosa of the guinea pig ileum. J. Neurocytol. 17, 451–463. Luff, S.E., McLachlan, E.M., 1989. Frequency of neuromuscular junctions on arteries of different dimensions in the rabbit, guinea pig and rat. Blood Vessels 26, 95–106. Luff, S.E., McLachlan, E.M., Hirst, G.D.S., 1987. An ultrastructural analysis of the sympathetic neuromuscular junctions on arterioles of the submucosa of the guinea pig ileum. J. Comp. Neurol. 257, 578–594. Luff, S.E., Hengstberger, S.G., McLachlan, E.M., Anderson, W.P., 1991. Two types of sympathetic axon innervating the juxtaglomerular arterioles of the rabbit and rat kidney differ structurally from those supplying other arteries. J. Neurocytol. 20, 781–795. Luff, S.E., Hengstberger, S.G., McLachlan, E.M., Anderson, W.P., 1992. Distribution of sympathetic neuroeffector junctions in the juxtaglomerular region of the rabbit kidney. J. Auton. Nerv. Syst. 40, 239–254. Luff, S.E., Young, S.B., McLachlan, E.M., 1995. The proportions and structure of contacting and non-contacting varicosities in the perivascular plexus of the rat tail artery. J. Comp. Neurol. 361, 699–709. Notoya, M., Nakamura, M., Mizojiri, K., 1996. Effects of lisinopril on the structure of renal arterioles. Hypertension 27, 364–370. Peachey, L., 1958. A study of section thickness and physical distortion produced during microtomy. J. Biophys. Biochem. Cytol. 4, 233–238. Purves, D., Voyvodic, J.T., Magrassi, L., Yawo, H., 1987. Nerve terminal remodelling visualized in living mice by repeated examination of the same neuron. Science 238, 1122–1126. Purves, D., Snider, W.D., Voyvodic, J.T., 1988. Trophic regulation of nerve cell morphology and innervation in the autonomic nervous system. Nature 336, 123–128. Reed, G., Moeller, I., Mendelsohn, F., Small, D., 1996. A novel action of angiotensin peptides in inhibiting neurite outgrowth from isolated chick sympathetic neurons in culture. Neurosci. Lett. 210, 209–212. Reinecke, M., Forssmann, W.G., 1988. Neuropeptide Žneuropeptide Y, neurotensin, vasoactive intestinal polypeptide, substance P, calcitonin gene-related peptide, somatostatin. immunohistochemistry and ultrastructure of renal nerves. Histochemistry 89, 1–9. Taugner, R., Hackenthal, E., 1989. The Juxtaglomerular Apparatus. Springer-Verlag, Berlin. Todd, M.E., 1986. Trophic interactions between rat nerves and blood vessels in denervated peripheral arteries and in anterior eye chamber transplants. Circ. Res. 58, 641–652. Wilkinson, L., 1990. SYSTAT: The System for Statistics. SYSTAT, Evanston.
Extended angiotensin converting enzyme inhibition changes the innervation of renal glomerular afferent arterioles Susan E. Luff ) , Simone B. Young, Warwick P. Anderson Department of Physiology, Monash UniÕersity, Wellington Road, Clayton, Victoria 3168, Australia Received 10 November 1998; received in revised form 21 May 1999; accepted 17 June 1999
Abstract Chronic inhibition of the angiotensin I converting enzyme ŽACE. with enalapril, results in a phenotypic change of the medial cells of renal afferent arterioles from contractile smooth muscle cells to renin containing epithelioid cells. In normal animals, the density of the innervation of the juxtaglomerular renin containing epithelioid cells is much lower compared to the contractile cells. The effector tissues are known to play an important role in determining the pattern and density of their innervation. In this study, we tested the hypothesis that the density of the innervation of the afferent arteriole smooth muscle cells decreases when they change their phenotype from contractile to renin containing epithelioid cells. The results show that the density of the innervation had significantly increased and the association of the terminals with the smooth muscle cells had changed. There were significantly more varicosities around renal afferent arterioles from rabbits treated with enalapril Ž10 mgrkgrh. for 6 weeks Žmean " SEM s 634 " 175 = 10 3rmm2 vessel surface, cf. 329 " 69 = 10 3rmm2 vessel surface in untreated rabbits, P s 0.05., with the number of neuroeffector junctions remaining the same Ž124 " 14 and 164 " 32 = 10 3rmm2 vessel surface. and significantly more non-contacting varicosities Ži.e. lying) 100 nm from the medial cells. Ž74 " 5% and 25 " 7%, respectively; P s 0.003.. Thus, there was no reduction in the innervation of afferent arterioles in which the smooth muscle cells had changed phenotype in response to enalapril treatment as hypothesised. Instead, it would appear that proliferation of the innervation had occurred, with the formation of additional varicosities but these varicosities failed to form neuromuscular junctions. This study has identified a form of neural plasticity in the kidney that has not previously been described. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Kidney; Blood vessels; Nerve growth; Plasticity
1. Introduction The density and form of the sympathetic innervation differs in different tissues. In arteries, the density of neuromuscular junctions Ži.e. ‘innervation density’, see Luff and McLachlan, 1989. is inversely proportional to the vessel diameter. In addition, the proportion of varicosities around vessels that form junctions is very high Ž70–92%. in the small arteriolar vessels ŽLuff et al., 1991. but is much lower in the larger muscular arteries Že.g. - 50% in rat tail artery, Luff et al., 1995. and in the larger elastic arteries, only non-contacting varicosities lying at variable distances
) Corresponding author. Tel.: q61-3-9905-1466; fax: q61-3-99052566; e-mail: [email protected]
from the smooth muscle cells have been reported ŽLuff and McLachlan, 1989.. What determines the innervation of different tissues is not understood although there is evidence that the tissue itself plays a major role ŽTodd, 1986.. Similarly, our knowledge of the plasticity of varicosities and neuroeffector connections of peripheral autonomic terminals on their targets of mature animals is limited. However, in vivo fluorescence studies of autonomic ganglia have demonstrated considerable changes occur in both the form of the dendritic arbours and the location of synaptic endings on the surface of identified autonomic ganglion cells when monitored over a period of several weeks ŽPurves et al., 1987.. The media of afferent arterioles from the outer cortical region of the rabbit kidney consists of a single layer of smooth muscle cells. Under normal circumstances, most of
0165-1838r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 1 8 3 8 Ž 9 9 . 0 0 0 4 7 - 8
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the cells along the length of the afferent arterioles are contractile smooth muscle cells but typically, they become epithelioid in nature at their distal end and contain large homogeneous electron dense granules of renin. In these cells, contractile elements with filament attachment sites and caveoli, typical of contractile smooth muscle cells, are much reduced or absent ŽTaugner and Hackenthal, 1989.. However, the number of renin containing epithelioid cells in the afferent arterioles may increase markedly during long term stimulation of the renin–angiotensin system, such as with angiotensin I converting enzyme ŽACE. inhibitors or low salt diets ŽIce et al., 1988; Barrett et al., 1989.. The increase in the number of renin secreting cells within the media of preglomerular arterioles results from the conversion of existing contractile smooth muscle cells to the renin containing epithelioid phenotype rather than cell division of the existing renin secreting cells ŽCantin et al., 1977.. In this study, we tested the hypothesis that the change in phenotype of the medial cells of afferent arterioles in response to enalapril treatment affects the innervation of this important renal vessel. The renal afferent arterioles receive a dense innervation predominantly by sympathetic postganglionic axons ŽBarajas and Muller, 1973; Gorgas, 1978; Reinecke and ¨ Forssmann, 1988; Barajas et al., 1992. and these nerves play an important role in regulating glomerular blood flow and hence, filtration rate as well as renin secretion ŽDiBona and Kopp, 1997.. Both the contractile smooth muscle and renin containing epithelioid cells, are innervated ŽBarajas and Muller, 1973; Gorgas, 1978. and physio¨ logical studies have shown that different receptor mechanisms operate post-junctionally in neurally mediated responses of contractile smooth muscle cells and renin containing epithelioid cells ŽDiBona and Kopp, 1997.. More recently, we have shown that renal afferent arterioles have a high density of neuromuscular junctions, it being one of the most densely innervated arteries so far studied ŽLuff and McLachlan, 1989; Luff et al., 1991.. In the normal rabbit kidney, neuromuscular junction density on contractile smooth muscle cells of afferent arterioles is up to 10 times greater than on renin secreting cells in the same vessel ŽLuff et al., 1992.. In this study, we have investigated whether the density of the innervation of the preglomerular afferent arterioles is markedly reduced Ži.e. from their normal high density to the lower density of juxtaglomerular epithelioid cells. when the phenotype of the medial cells is changed from contractile smooth muscle cells to renin containing epithelioid cells. We have used ACE inhibition by administering enalapril continuously for 6 weeks to promote the phenotypic change of these cells. Quantitative ultrastructural techniques developed by us ŽLuff et al., 1992., were employed to compare the innervation of afferent arterioles from control untreated rabbits with those of treated rabbits in which the most of the medial cells had changed to the renin secretory phenotype.
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2. Materials and methods 2.1. Tissue preparation Nine male rabbits aged 20–25 weeks were allocated to either enalapril-treated Ž n s 4. or untreated Ž n s 5. groups. In the first group, enalapril was administered subcutaneously via mini-pumps ŽAlza, Palo Alto, CA, USA. implanted under the skin between the scapulae under local anaesthesia ŽLignocaine, Delta West, Western Australia.. Mini-pumps were changed at 4 weeks to ensure a continuous infusion of enalapril ŽMaleate salt. of 10 mgrkgrh for 6 weeks. At 6 weeks, all rabbits were anaesthetized with pentobarbitone Ž60 mgrkg, i.v.. and prepared for perfusion fixation of the kidneys using the method described previously ŽLuff et al., 1987.. Catheters were inserted in the central ear artery and vein to measure arterial blood pressure responses to a bolus intravenous injection of angiotensin I ŽAng I. Ž10 ngrkg for untreated control animals and 100 ngrkg for treated animals. to establish the extent of ACE inhibition in enalapril-treated rabbits compared to untreated control rabbits. In all animals, the blood pressure was allowed to return to pre-injection levels. A bolus injection of 3 ml of 1% sodium nitrite plus 5000 IU of heparin was then given via the ear vein to dilate the vasculature. The kidneys were immediately perfused via the abdominal aorta with a fixative solution containing 2% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer at pH 7.3 at a pressure of 130 mm Hg for approximately 10 min. The kidneys were removed and tissue from the outer cortical region was cut into small pieces Žapproximately 2 mm wide, 3 mm long and 1 mm thick. and fixed at room temperature for a further 2 h in the same fixative. The tissue was then washed in buffer and postfixed in buffered 2% osmium tetroxide for 4 h, dehydrated and embedded in epoxy resin as described previously ŽLuff and McLachlan, 1988.. 2.2. Tissue sectioning and sampling Thin Ž1 mm thick. sections were prepared from blocks of outer cortical tissue to locate afferent arterioles cut in longitudinal section along their entire length Ži.e. from the origin with the interlobular Žcortical radial. artery to the connection with the glomerulus. ŽFig. 1.. Similarly oriented afferent arterioles were selected for ultra microtomy from untreated and enalapril-treated rabbits, but in addition, enalapril-treated afferent arterioles were selected that had most Žmean " SEMs 93 " 4%. of the medial cells packed with renin secretory granules ŽFig. 1B., which was confirmed in low power electron micrographs Žmagnification s =2000; see Fig. 1B.. These selection criteria maximised the vessel adventitial surface present in each section, reducing the number of sections required for each analysis.
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Fig. 1. Low power electron micrographs of two afferent arterioles cut in longitudinal section. ŽA. An afferent arteriole from a control rabbit with the media largely consisting of contractile smooth muscle cells. ŽB. An afferent arteriole from an enalapril-treated rabbit with the media largely consisting of renin containing epithelioid cells. dt, Distal tubule; g, glomerulus; I, intralobular artery; l, lumen of afferent arteriole; pt, proximal tubule. Calibration bar in B s 20 mm and also applies to A.
Ultra-thin sections Ž; 100 nm thick. were prepared from selected enalapril-treated and untreated afferent arterioles in one of two ways: Ži. short ribbons of ; 10 sections, taken every 2 m Ži.e. four to six ribbons of sections per afferent arteriole. from nine enalapril-treated and nine untreated afferent arterioles, Žii. serial sections cut through 2.3–8.7 mm of tissue Ži.e. 23–87 sections. from the mid region of each of four untreated and three enalapril-treated afferent arterioles obtained from three rabbits in each case. Section thickness was determined from the interference colour of individual sections floating on water ŽPeachey, 1958. recorded at the time of cutting Ži.e. pale gold s approximately 100 nm. and used to calculate the surface area analysed in each section Žsee below.. A previous analysis of rabbit afferent arterioles had shown that the size of varicosities ranged from 0.25 to 1.23 mm Žsee Table 1, Luff et al., 1991. hence, a separation of ) 2
mm between sections in Ži. above was chosen to ensure that the same varicosity was not measured twice. 2.3. Analysis Varicosities and neuromuscularrneuroeffector junctions were defined as follows: Õaricosities are axon profiles G 0.25 mm in diameter that contained small Ž50 nm. synaptic vesicles ŽFigs. 2 and 3.; neuromuscular r neuroeffector junctions are varicosities in contact with either a vascular smooth muscle cell Žneuromuscular. or renin containing epithelioid cell Žneuroeffector., with the intervening gap being - 100 nm and containing a single basal lamina Žsee Luff et al., 1991. ŽFig. 2.. Axons were identified as associated with afferent arterioles if they were bare of their Schwann cell along their surface facing the arteriole and lay - 1 mm from an arteriole. These criteria resulted from previous studies on the innervation of the
S.E. Luff et al.r Journal of the Autonomic NerÕous System 77 (1999) 114–124 Table 1 Vessel diameter, vessel length and vessel surface area per vessel length of afferent arterioles from enalapril-treated and untreated rabbits Treatment
Diameter Žmm.
Vessel length Žmm.
Vessel surface arearmm length of vessel Žmm2 rmm.
Control vessels Enalapril-treated vessels P values
20.8"2.5, ns10 33.0"8.6, ns10
121.6, ns9 101.8, ns9
33.0, ns 4 30.8, ns 4
0.001
0.55
0.63
Measurements of vessel surface arearunit length of vessel were taken from sections that were cut through the mid region of the vessel through the point where they entered the glomerulus and connected with the interlobular artery.
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outer cortical arterioles showing that most Ž) 70%. varicosities formed neuroeffector junctions and lay - 1 mm from the cells that they eventually contacted ŽLuff et al., 1991.. In cases where arterioles lay - 1 mm from a tubule, adjacent sections were used to establish whether the synaptic vesicles were clustered towards the afferent arteriole or the adjacent tubule. 2.3.1. Measurement of the proportion of Õaricosities that formed junctions Serial thin sections were used for these analyses. A total medio-adventitial surface area ranging from 1190 to 2520 mm2 Žmean " SEM s 2134 " 821 mm2 , n s 3. for enalapril-treated and 436–2442 mm2 Žmean " SEM s
Fig. 2. Electron micrographs of two sympathetic neuroeffector junctions. ŽA. On a contractile smooth muscle cell of a control afferent arteriole and ŽB. on a smooth muscle cell that has converted to a renin containing epithelioid cell of an afferent arteriole from an enalapril-treated rabbit. In both cases, the varicosities have extended regions of their membrane apposed to the effector cell membrane with the intervening gap being F 100 nm and containing a single basal lamina. Note that the cytoplasm of the smooth muscle cells in A, typically have dense bands and dense bodies and contractile filaments throughout the cell, whereas the epithelioid cell in B, is packed with large electron dense renin granules, endoplasmic reticulum and ribosomes, the contractile filaments being reduced and restricted to a region just under the cell membrane. rc, Renin containing epithelioid cell; sm, contractile smooth muscle cell; v, varicosity. Calibration bar in B s 1 mm and also applies to A.
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Fig. 3. Electron micrographs of non-contacting varicosities. ŽA. An example of a non-contacting varicosity from an untreated afferent arteriole. It is typically small, lacks a mitochondrion and does not have any obvious clustering of vesicles normally associated with vesicle exocytosis. ŽB. A large non-contacting varicosity from an enalapril-treated afferent arteriole which has a clustering arrangement of synaptic vesicles associated with electron dense thickenings of the varicosity membrane Žarrows. typical of a presynaptic membrane specialisation ŽPMS. associated with vesicle exocytosis. Calibration bar in B s 1 mm and also applies to A. rc, Renin containing cell, rg, renin granule, sm, smooth muscle cell, v, varicosity.
1211 " 509 mm2 , n s 4. for untreated afferent arteriole was analysed. The combined area for all the afferent arterioles analysed from the enalapril-treated and untreated group was 6401 mm2 and 4843 mm2 , respectively. Initially, the adventitia of the afferent arterioles in the mid section of each series of sections was scanned in the electron microscope at 5000 times magnification and all the axon profiles located and photographed. These micrographs were used to locate the axon profiles in each section within the series which were subsequently photographed at a higher Ž=15 000. magnification to produce sets of micrographs for complete varicosities. These sets of micrographs were used to determine the proportion of varicosities around each vessel that formed junctions. 2.3.2. Measurement of Õaricosity Õolume and neuromuscular r neuroeffector contact area Measurements of varicosity volume and junction size were obtained from sets of micrographs obtained from serial sections Žas detailed above. of 14 contacting varicosities randomly selected from three enalapril-treated and three untreated afferent arterioles each from a separate animal, using a graphics tablet and custom computer software ŽLuff et al., 1987.. Similar measurements were made of 24 and 8 non-contacting varicosities around enalapriltreated and untreated afferent arterioles respectively, to compare their size in each group of vessels. 2.3.3. NerÕe density measurements In this study, we will refer to density values for varicosities obtained using single sections, as varicosity density. We have previously defined the density of junctions measured in single sections that are expressed as the
number of junctions per smooth muscle cell surface area analysed, as ‘innervation density’ Žsee Luff and McLachlan, 1989.. In contrast, measurements of the number of junctions per smooth muscle cell surface area analysed in serial sections of tissue containing complete varicosities, is true ‘junction density’. We have consequently retained these definitions. The surface area analysed for each afferent arteriole was obtained by tracing around the outer surface of the smooth muscle cells of each vessel profile in low power Žmagnifications 600 times. electron micrographs using a graphics tablet and custom computer software. The medio-adventitial length obtained was subsequently multiplied by the section thickness to obtain the surface area analysed in each section. The number of varicosities and neuromuscular and neuroeffector junctions was obtained from four to six single sections Ž) 2 mm apart. of nine control untreated and nine enalapril-treated afferent arterioles from four and three rabbits, respectively. The mean surface area of vessels analysed per rabbit were: control rabbitss 189 and 94 mm2 Ž n s 3 vessels. from 2 rabbits respectively, 300 mm2 Ž n s 2 vessels. from a third rabbit and 103 mm2 of a single vessel from a fourth rabbit; enalapril-treated vesselss 149, 126, and 113 mm2 Ž n s 3 vessels from each rabbit.. The individual data obtained from each section was combined for each afferent arteriole and the density values expressed as the number of varicosities or junctions per mm2 of vessel surface area. 2.3.4. Afferent arteriole measurements To determine whether enalapril treatment led to changes in diameter and or surface area of afferent arterioles, the outer vessel dimensions were measured from transverse
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sections of 10 afferent arterioles in tissue from two enalapril-treated and two untreated rabbits. The mean diameter of each afferent arteriole was determined from measurements of their maximum and minimum diameters, and the outer surface of the smooth muscle cells at the medio-adventitial border of the arteriole in each section was measured using a graphics tablet and computer software. Additional measurements were made of vessel length, measured in longitudinal sections from the mid region of afferent arterioles Žfour control and four enalapril-treated. down the centre of the lumen from the point where the vessel enters the glomerulus and its point of branching off the interlobular artery. In the same section, we measured along the outer surface of the smooth muscle cells at the medio-adventitial surface. From these two measurements, we calculated the outer cell surface length per unit vessel length to establish whether the change in cell shape had contributed to the change in medio-adventitial surface area of the vessel. 2.3.5. Statistics The data are presented as means " standard error of the mean. A one-way analysis of variance was used to test the statistical significance between values obtained for enalapril-treated and untreated groups of afferent arterioles. P values F 0.05 were considered to be significantly different. Relations between junction density and afferent arteriole diameter were tested using linear regression analysis. All calculations were performed using Statview ŽAbacus Concepts, Berkeley, CA, USA. and SYSTAT ŽWilkinson, 1990. software.
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These experiments were conducted in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. 3. Results The mean arterial pressure was similar for the treated and untreated anaesthetized rabbits Žrange 80–110 mm Hg and 87–94 mm Hg, respectively.. There was no discernible effect on blood pressure following a bolus injection of Ang I of 100 ngrkg in enalapril-treated animals. In contrast, 10 ngrkg Ang I in the untreated control rabbits, increased mean arterial blood pressure by G 15 mm Hg. 3.1. General morphology of outer cortical afferent arterioles Renin secreting cells in the media of arterioles were easily identified in 1 mm thick sections stained with toluidine blue when viewed in the light microscope at =1000 magnification, by the presence of dark blue staining renin granules. In tissue from untreated rabbits, the media of afferent arterioles consisted almost entirely of a single layer of smooth muscle cells of the contractile phenotype with only the one to two cells adjacent to the glomerulus containing renin granules ŽFig. 4.. In tissue from enalapril-treated rabbits, the conversion of contractile smooth muscle cells to the renin containing epithelioid phenotype occurred in the preglomerular rather than the post-glomerular blood vessels, with the afferent arterioles being the ones predominantly affected. However, renin granules were also observed in the medial cells of some
Fig. 4. Electron micrograph of an afferent arteriole from a control rabbit at the region where it connects with the glomerulus showing contractile smooth muscle cells Žsm. and renin containing epithelioid cells Žrs.. g, Glomerulus; l, lumen of afferent arteriole; pt, proximal tubule; dt, distal tubule. Calibration bar s 1 mm.
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interlobular arteries. In the nine afferent arterioles selected for analysis from enalapril-treated rabbits, 93 " 4% of cells contained renin granules Ži.e. 100% of the medial cells in five afferent arterioles, 95% in two, and 85% and 70% in each of two other afferent arterioles.. The cells that did not contain renin granules in these vessels lay closer to the interlobular artery ŽFig. 1B.. 3.2. Vessel dimensions In all the afferent arterioles analysed from enalapriltreated animals, it was notable that they appeared to have greater outside and lumina diameters than those from control untreated animals Žcf. Fig. 1A and B.. Also, the renin secreting cells appeared larger in cross-section compared to the smooth muscle cells of the afferent arterioles from untreated rabbits ŽFig. 1.. The outer diameters of afferent arterioles measured in transverse sections were significantly greater for enalapril-treated compared to untreated control vessels Ž P s 0.001. ŽTable 1.. Similarly, the vessel perimeter measured along the outer surface of the smooth muscle cells in transverse sections was also significantly greater Ž P s 0.003.. The ratio of mean vessel diameter for untreated to enalapril-treated afferent arterioles was 0.64. This value was almost identical to that for the ratio of the vessel perimeter measured in transverse section of afferent arterioles from untreated to enalapriltreated afferent arterioles measured in transverse section Ži.e. 0.63.. Measurements of vessel length Žobtained from longitudinal sections. showed that there was no significant difference between the enalapril-treated and untreated groups of afferent arterioles Žmean s 101.8 and 121.6, respectively, P s 0.55.. Also, there was no significant difference in smooth muscle cell surface length per unit vessel length measured in longitudinal sections of enalapril-treated and untreated groups of vessels. Hence, the increase in vessel surface area in the enalapril-treated afferent arterioles can be predominantly attributed to the increase in vessel diameter. Consequently, it was necessary to take into account these differences in vessel diameter to adjust the axon terminal density measurements obtained for the enalapriltreated group of vessels Žsee below.. 3.3. InnerÕation analysis Junctions were observed on both types of cells, i.e. smooth muscle cells in control afferent arterioles and renin secreting cells in afferent arterioles from enalapril-treated tissue. There was no apparent difference in the structure of the neuroeffector junctions Žcf. Fig. 2A and B.; all contained small synaptic vesicles with some having obvious aggregations towards the region of membrane contact ŽFig. 2B.. Also, a similar proportion of junctions Ž22 " 2% in enalapril-treated and 25 " 6% from untreated afferent arterioles. had presynaptic membrane specialisations ŽFig. 2..
At the regions of contact between the nerve varicosities and either the smooth muscle or renin containing epithelioid cells, the gaps between the cell membranes were - 100 nm wide and contained a single basal lamina. In the afferent arterioles from untreated rabbits, the non-contacting varicosities were small Ž- 1 mm in diameter. ŽFig. 3A. and although they contained synaptic vesicles, none had presynaptic membrane specialisations. Conversely, large Ž) 1 mm in diameter. non-contacting varicosities were observed around the enalapril-treated afferent arterioles and some Ž11%. did have presynaptic membrane specialisations ŽFig. 3B.. Most varicosities contained large granular vesicles. It was noticeable that small Ž50 nm. granular vesicles were present in some individual profiles of varicosities whereas others contained only agranular vesicles Žsee Figs. 2 and 3.. However, further investigation of varicosities in serial sections revealed that although individual profiles only contain agranular vesicles, other profiles of the same varicosity contain granular vesicles. Also, individual axons sometimes had varicosities with only agranular vesicles, and others that contained granular vesicles. We therefore concluded that the presence of granular vesicles was not a suitable criteria for distinguishing nerve types. Data obtained from 6-hydroxydopamine treated tissue from the outer cortex of an enalapril and an untreated rabbit, demonstrated that all the varicosities associated with the afferent arterioles Ž90 varicosities from three different pieces of tissue from each animal. reacted positively, indicating that they were all catecholaminergic. In addition, no negatively reacting varicosities were observed associated with any of the other vessels in the same tissues. 3.3.1. Measurements of Õaricosity size and neuromuscular r neuroeffector contact areas The varicosity volumes and neuroeffector contact areas of contacting varicosities in enalapril-treated and untreated afferent arterioles ranged from 0.12 to 1.79 mm3 and 0.02–1.03 mm3 ; 0.03–1.04 mm2 and 0.02–0.89 mm2 , respectively ŽFig. 5.. These values were not significantly different Ž P s 0.11 and 0.16, respectively.. Conversely, the non-contacting varicosities from enalapril-treated animals were significantly larger than those around the untreated afferent arterioles Žrange s 0.05–2.21 mm3 ; n s 24 and 0.04–0.44 mm3 n s 8, respectively; P s 0.003.. 3.3.2. Density of junctions and Õaricosities The number of varicosities analysed in each afferent arteriole ranged from 19 to 74 Žmean " SEM for enalapril-treated afferent arterioless 44 " 7 and for untreated afferent arterioless 46 " 7.. The total number of varicosities analysed in the enalapril-treated and untreated group of afferent arterioles was similar Ž397 and 413, respectively.. The data obtained for the number of varicosities lying - 1 mm from the surface of afferent arterioles from
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Fig. 5. Histograms of mean values for ŽA. varicosity volume and ŽB. neuromuscular contact area of varicosities innervating afferent arterioles from enalapril-treated and untreated rabbits.
enalapril-treated rabbits, before adjustment for the differences in vessel dimensions Žsee Section 3.2., was 401 " 97 = 10 3rmm 2 of vessel surface and untreated rabbits was 329 " 69 = 10 3rmm2 of vessel surface Ž P s 0.53., with the number of junctions on smooth muscle cells of afferent arterioles from enalapril-treated and untreated rabbits being 84 " 13 and 164 " 32 = 10 3rmm 2 of vessel surface, respectively Ž P s 0.025.. The relationship between the log 10 transformation of ‘innervation density’ measurements Ži.e. the number junctions per mm2 of vessel surface measured using single section analysis. and afferent arteriole diameter was linear Ž r s 0.71; P - 0.0001. ŽFig. 6.. This data together with the data obtained for vessel dimensions Žsee above. demonstrate the necessity to adjust the density measurements for varicosities and neuroeffector junctions in enalapril-treated afferent arterioles for the difference between vessel diameters by multiplying the values for each vessel by the ratio of the mean diameters of afferent arterioles of untreated to enalapril-treated rabbits ŽFig. 7.. The data showed that varicosity density was greater in enalapril-treated rabbits Ž329 " 69 and 634 "
Fig. 6. Linear regressions of the log 10 transformed ‘innervation density’ Žexpressed as number of junctions per mm2 of afferent arteriole vessel surface. and afferent arteriole diameter.
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Fig. 7. Histograms of ŽA. varicosity density and ŽB. innervation density, after adjustment for differences in afferent arteriole diameter. Density values are expressed as number of junctions or varicosities per mm2 of afferent arteriole vessel surface.
175 = 10 3rmm2 of afferent arteriole surface, P s 0.05. whereas the ‘innervation density’ for afferent arterioles from untreated and enalapril-treated rabbits were not significantly different Žmean s 164 " 32 and 124 " 14 = 10 3rmm 2 of afferent arteriole surface, respectively, P s 0.24.. 3.3.3. Measurements of the proportion of total number of Õaricosities that form junctions Measurement of the proportion of varicosities that formed neuroeffector junctions was calculated using serial section analysis of three afferent arterioles Žeach from a different animal. from enalapril-treated rabbits and from four afferent arterioles Žeach from a separate animal. from untreated rabbits. In the afferent arterioles from untreated rabbits, most varicosities Ž75 " 5%. formed junctions with contractile smooth muscle cells of the media. However, the proportion of varicosities that formed junctions with renin secreting cells of the media of afferent arterioles from enalapril-treated rabbits was significantly lower Žmean s 26 " 5%; P s 0.003. ŽFig. 8..
Fig. 8. Histogram of the percentage of varicosities associated with afferent arterioles that form junctions in vessels from enalapril-treated and control untreated animals.
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4. Discussion Extended treatment of rabbits with the angiotensin converting enzyme inhibitor enalapril resulted in changes in the phenotype of many of the vascular smooth muscle cells of renal afferent arterioles Žsee also Barrett et al., 1989., with the media of some vessels consisting entirely of renin containing epithelioid cells. In afferent arterioles selected for study from the outer cortical region of the kidney, ) 90% of the contractile smooth muscle cells had changed their phenotype to renin containing epithelioid cells that contained large electron dense renin granules. The detailed ultrastructural analysis conducted on the innervation of the afferent arterioles in this study encompassed a number of different aspects to ascertain whether there was any quantitative and or structural change to the innervation in the enalapril-treated afferent arterioles compared to control untreated vessels; these included, the structure of the terminals and their relationship with the smooth muscle cells as well as the density of varicosities and neuroeffector junctions around the afferent arterioles. The data clearly demonstrate that the axon terminals innervating afferent arterioles from enalapril-treated rabbits differed structurally, quantitatively and in their relationship with smooth muscle cells from those innervating afferent arterioles from control untreated rabbits. 4.1. Structure of Õaricosities and their association with smooth muscle cells There was a marked difference between the afferent arterioles of enalapril-treated and control animals, in the proportion of varicosities around the vessels that had formed junctions, the proportion being considerably lower Ž26%. for the enalapril-treated compared to the untreated vessels Ž75%.. The proportion of varicosities forming junctions in the control Žuntreated. arterioles was similar to that reported in a previous study Ži.e. 70–93%; see Luff et al., 1991.. This analysis was conducted on serial sections through entire varicosities and consequently, is independent of varicosity size, neuroeffector contact area size and is not related in anyway to the size of the vessel. There was also a difference in the structure of the non-contacting varicosities innervating the afferent arterioles from enalapril-treated rabbits. In the afferent arterioles from untreated rabbits in the current study as well as our previous study ŽLuff et al., 1991., it was notable that the varicosities that did not form junctions were small Ž- 1 mm in diameter., lacked mitochondria and contained only a few synaptic vesicles. Conversely, in the afferent arterioles from enalapril-treated rabbits there were many noncontacting varicosities that were large Ž) 1 mm in diameter. and contained numerous synaptic vesicles, with some having presynaptic membrane specialisations similar to those found around larger muscular arteries ŽLuff et al., 1995.. This suggests that neurotransmitter release may be
occurring at both junctional and extra-junctional sites in the afferent arterioles from enalapril-treated rabbits but is predominantly released at junctions in vessels from control rabbits. 4.2. Density of Õaricosities and neuroeffector junctions The density of varicose terminals around afferent arterioles of enalapril-treated rabbits was significantly greater compared to that around control vessels. In contrast, the ‘innervation density’ values Ži.e. number of neuroeffector junctions per mm2 of vessel surface. were similar for both groups of afferent arterioles. These ‘innervation density’ values were not related to changes in the size of the terminals or the neuroeffector contact areas, as neither of these values differed for the two groups of afferent arterioles. The density values for neuromuscular junctions obtained from analysis of single sections of vessels, is not the true junction density and is greater than the density values obtained from serial sections previously published. In this previous study, we showed that ‘innervation density’ for renal afferent arterioles are approximately eight times greater than junction density values obtained from analyses conducted in serial sections ŽLuff et al., 1992.. Measurements of ‘innervation density’ reflects the frequency of junctions encountered in a single section, whereas measurements in serial sections represents true junction density. The difference in the values is related to the size of the junctions and number of sections in which the junctions fall Žsee Luff et al., 1992.. We previously demonstrated that the density of neuromuscular junctions on afferent arterioles from normal untreated rabbits is much lower Ž; 10%. on the renin secreting cells than on the contractile smooth muscle cells in the same vessel Žmean s 2.25 and 20.0 = 1000rmm2 of cell adventitial surface, respectively ŽLuff et al., 1992... Hence, we predicted that a reduction in the density of junctions would result with the change of cell phenotype resulting from enalapril treatment. Such a result would be in agreement with other studies where the pattern of innervation, including that of blood vessels ŽTodd, 1986. and salivary glands ŽPurves et al., 1988. is regulated by the effector tissue. However, the data indicates that the innervation had proliferated rather than reduced with an increase in the number of non-contacting varicosities but not in the varicosities forming neuroeffector junctions. This is supported by the data obtained from serial sections for the proportion of varicosities forming junctions Žwhich are independent of changes in vessel surface area. showing a significantly greater number of non-contacting varicosities around the enalapril-treated group of afferent arterioles. Hence, it would appear that the phenotype of the cells forming the media of renal afferent arterioles is not important in determining density of axon terminals innervating these vessels at least, after the innervation has been established. Alternatively, autonomic neuromuscular junctions may be stable structures once they have formed. Our knowledge of the
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plasticity of varicosities and neuroeffector connections of peripheral autonomic terminals on their targets of mature animals is limited. Nevertheless, it has been demonstrated in vivo that the distribution of presynaptic endings on the surface of autonomic ganglion cells changes over a period of 1–3 weeks ŽPurves et al., 1987. indicating that autonomic nerves can and do change their synaptic connections at least in ganglia. Further studies on the innervation of afferent arterioles in tissue from enalapril-treated animals that did not exhibit any phenotypic change in their smooth muscle cells would provide information on whether the cell phenotype is important in promoting the sprouting of varicosities and preventing the formation of additional neuroeffector junctions observed in this study. We interpret the results as indicating that varicosities that were originally in contact with contractile smooth muscle cells have remained in contact with them after they have changed phenotype. Furthermore, that new varicosities had developed around the enalapril-treated group of vessels but had not formed junctions with the cells of altered phenotype. This gives rise to the question: why have new varicosities formed? Neurite outgrowth from isolated chick sympathetic neurons has been shown to be inhibited in the presence of Angiotensin II ŽAng II. ŽReed et al., 1996.. Hence, an explanation for the increase in number of varicosities around the enalapril-treated afferent arterioles may lie in the abnormally low level of circulating Ang II resulting from ACE inhibition. The role of Ang II in the growth of nerves in vivo is not fully understood and certainly during development, the opposite case is true in that Ang II promotes nerve growth factor release, which in turn promotes the growth of nerves ŽAlcorn et al., 1996.. However, the receptors involved in this interaction during development are AT2 receptors, which are not normally present in this tissue in adult animals ŽAlcorn et al., 1996.. An explanation for why the newly formed varicosities did not form junctions with smooth muscle cells that have converted to renin containing epithelioid cells could be that the cells with established neuroeffector junctions have rejected axons forming any further neuroeffector contacts. Evidence for this comes from a study of the growth of sympathetic neurons together with smooth muscle cells in culture ŽChamley et al., 1973., which showed that if more than one axon contacted the same smooth muscle cell at approximately the same time, then all axons would form lasting contacts. Conversely, axons that formed contacts with smooth muscle cells that already had established neuromuscular junctions, failed to form lasting contacts. Another possible explanation is that the 6-week treatment period was insufficient for newly formed varicosities around the enalapril-treated afferent arterioles to form neuroeffector junctions, but this is unlikely as during early postnatal life, axon varicosities form and establish neuromuscular junctions on rat mesenteric arteries within 4–8 days ŽLuff, 1999..
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4.3. Adjustment of density measurements for differences in Õessel dimensions The results of this study clearly show that there is a change in the innervation of afferent arterioles in response to enalapril treatment and that this is true whether the data is corrected for the increase in vessel surface area or not. The question being addressed was, ‘‘whether the number of junctions become reduced in response to enalapril treatment in vessels where the smooth muscle cells have changed their phenotype.’’ To quantify this, we have measured ‘innervation density’ which is expressed as the number of junctions per unit area of the outer surface of the smooth muscle cells at the medio-adventitial border. Prior to enalapril treatment, each vessel would have started with a finite number of axon varicosities and neuromuscular junctions. As a result of enalapril treatment, it is evident both from our own studies and others Že.g. Notoya et al., 1996., there is a significant increase in the diameter Žboth internal and external. of afferent arterioles from kidneys that have been subjected to ACE inhibition over a period of several weeks resulting in an increase in the surface area of the vessel at the medio-adventitial border where the nerve terminals are located. Hence, if there is no change in the number of terminals or junctions around each vessel in response to enalapril treatment then because the surface area of the vessels is increased, the density of both junctions and varicosities would be lower than that on the control vessels. This was not the case; the junction density was lower but the varicosity density was unchanged. In order to establish and quantify what had changed and by how much, it was necessary to correct the density measurements for the change in the outer surface area of the smooth muscle cells. We have shown that the outer surface of the smooth muscle cells did change in response to an increase in vessel diameter but did not change due to a change in cell shape or any restructuring of the vessel wall that might have occurred. This was demonstrated by the lack of change in either vessel length or in the cell surface lengthrunit vessel length measured in longitudinal sections through the mid region of the vessel. 4.4. Type of nerÕes The innervation of the cortical vessels is predominantly made up of sympathetic axons ŽGorgas, 1978; Reinecke and Forssmann, 1988; Barajas et al., 1992. but a small population of afferent nerves have also been demonstrated in a number of different animal species. In addition, there is evidence for heterogeneity of the sympathetic component of the innervation both with respect to their peptide content ŽReinecke and Forssmann, 1988; Knight et al., 1989. and structure ŽLuff et al., 1991.. Data obtained from vessels from animals pretreated with 6-hydroxydopamine indicates that the changes to the innervation demonstrated in this study predominantly if not exclusively involve the sympathetic nerves as in both the enalapril-treated and
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untreated tissues, all the axon terminals identified reacted positively with 6-hydroxydopamine Ži.e. contained small granular vesicles.. It is possible that a specific sub population of sympathetic nerves may be involved in the innervation changes demonstrated in this study, but to determine if this was the case, would required extensive investigation which is beyond the scope of this study.
5. Conclusion We have demonstrated a change in the innervation of rabbit afferent arterioles in which the majority of the contractile smooth muscle cells have converted to a renin containing epithelioid phenotype in response to 6 weeks treatment with enalapril. As a result of the enalapril treatment, the innervation of the afferent arterioles in which most of the medial cells were transformed to renin containing epithelioid cells, had altered. In particular, the proportion of total number of varicosities around these vessels that formed neuroeffector junctions was much lower. However, the results of this study did not support our original hypothesis that ‘innervation density’ would be reduced as a result of the change in medial cell phenotype. In fact, the data indicated that there was an increase in the innervation in response to enalapril treatment with more varicosities being formed, but the density of junctions remained the same as that on the untreated vessels. We hypothesise that the newly formed varicosities, failed to form neuroeffector junctions with the renin containing epithelioid medial cells. This study has identified a form of neural plasticity that has not been previously described. The increase in the density of the innervation in response to enalapril treatment is likely to have significant functional effects on renin release which remains to be determined.
Acknowledgements This work was supported by the National Heart Foundation of Australia and the National Health and Medical Research Council of Australia.
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