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Comparisons of prostaglandin vasoactive effects and interactions in the in vivo microcirculation of the rat urinary bladder
MICROVASCULAR
RESEARCH
17, I-11 (1979)
Comparisons of Prostaglandin Vasoactive Effects and Interactions in the in Viva Microcirculation of the Rat Urinary Bladder’ WILLIAM
F. YOUNG,JR.,
RICHARD D. DEY, AND ROBERT ECHT
Department of Anatomy, Michigan State University, Received
College of Human Medicine, East Lansing, Michigan 48824 May 26, 1978
A combination of techniques for in vivo transillumination, topical application of vasoactive agents, and direct microscopic observation of microcirculatory responses was utilized to evaluate the vasomotor actions of prostaglandins (PGs) E,, E,, F,,, F,, A,, and A, on rat urinary bladder arterioles and venules. The effects of PGE, and histamine (HIS) on arteriolar responsiveness to norepinephrine (NE), serotonin (5HT), and PGF, were measured. Histochemical studies were completed to determine the primary site of prostaglandin (PG) metabolic deactivation in the urinary bladder. Arteriolar dilatation occurred with HIS, PGE,, PGE,, PGA,, PGAf, and PGF,,, all of which (with the exception of HIS) demonstrated significant dose-related responses. Overall, PGE, and PGEZ were of greatest potency. Significant dose-related arteriolar constriction occurred with NE > PGF, > 5-HT (in order of decreasing potency). HIS, PGE,, PGE,, and PGA, produced significant venular dilatation; PGE, and HIS were dose related. Only NE resulted in significant venoconstriction. Arteriolar responsiveness to NE and PGF,, decreased after pretreatment with PGE, but was unchanged by HIS pretreatment, whereas application of 5-HT following pretreatment with PGE, or HIS produced equivalent levels of arteriolar constriction. The primary site of deactivation of PGE, was histochemically localized to bundles of smooth muscle fibers in the muscular coat of the rat urinary bladder wall.
INTRODUCTION Several previous investigations have provided models for defining the in viva vascular responses to exogenous prostaglandins (PGs).~ The most direct in vivo method of experimentation consists of local applications of the PGs and direct observations of their effects on the microcirculation (Kaley and Weiner, 1968; Weiner and Kaley, 1969; Siggins, 1972; Messinaer af., 1974). In this investigation, a combination of techniques for in viva transillumination, topical applications of vasoactive agents, and direct microscopic observations of the rat urinary bladder microvasculature was utilized to evaluate: (1) the vasomotor actions and relative potencies of norepinephrine (NE), serotonin (5-HT), histamine (HIS), and PGs El, E,, F1,, FZu, A,, and A,; and (2) the pretreatment effect of PGE, and HIS on the arteriolar responsiveness to PGF,,, NE, and 5-HT. In addition, histochemical * This study was supported in part by a grant from the Michigan Heart Association. Send reprint requests to Dr. Robert Echt, Department of Anatomy, College of Human Medicine, Michigan State University, East Lansing, Michigan 48824. L Abbreviations used: PGs, prostaglandins; PG, prostaglandin; NE, norepinephrine; 5-HT, serotonin, 5-hydroxytryptamine; HIS, histamine; 15-OH PGDH, 15-hydroxy prostaglandin.dehydrogenase; cyclic AMP, adenosine 3’, 5’-monophosphate; cyclic GMP, guanosine 3’, 5’-monophosphate. 1 00262862/79/01O~WCt-11$02.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
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studies were also completed to determine the primary site of PG metabolic deactivation in the rat urinary bladder. MATERIALS
AND METHODS
Anesthesia of male and female mature rats (200-325 g) was induced with an intraperitoneal injection of sodium pentobarbital (Jen-Sal), 30-50 mg/kg, and maintained with supplementary subcutaneous doses of 6 mg/kg every 20 min. A tracheotomy permitted adequate ventilatory exchange and decreased the body movement secondary to respiratory efforts. A ventral, abdominal, midline incision was made superior to the urinary bladder, which was then exposed with the peritoneum intact. The ureters were severed to ensure static bladder size and intraviscus pressure. The inferior and superior surfaces of the urinary bladder were continuously superfused with warmed (37.2”) mammalian Ringer’s solution flowing at a constant rate of 2 mllmin. The pH of the Ringer’s solution was adjusted to 7.4 with dry sodium bicarbonate. In four control animals, an indwelling bladder cannula was connected to a low-pressure transducer (Statham P23BB) to cystometrically monitor intrabladder pressures (Pitts, 1968). As warmed Ringer’s solution was infused in OS-ml increments, stable internal bladder pressures (11.5-14.0 mm Hg) were recorded between bladder volumes of 0.5 and 1.5 ml. Transverse and longitudinal bladder diameters were then measured with a caliper and correlated with bladder volumes. In all subsequent animals, repeated diameter measurements were made to ensure bladder volumes within the standardized range for consistent internal pressures. The apparatus first described by Knisely (1936) and recently modified by Wilson (1970) and Echt et al. (1976) was utilized for in vivo transillumination of the urinary bladder. Copper-constantan 25-gauge thermocouples were used for continuous monitoring of superior dripper, borosilicate glass rod tip (source of illumination and inferior super-fusion), and rectal temperatures. Urinary bladder surface temperatures were intermittently monitored with 40-gauge thermocouples. Body temperature was maintained in a cylindrical heating chamber made from tin and wrapped with electrical tape (Smith-Gates Corp.). All temperatures were recorded on a Grass polygraph and maintained at 37.2 + 0.5”. PG (Upjohn) stock solutions were prepared by dissolving 1.OOmg of each PG in 0.1 ml of absolute ethanol and then adding 0.9 ml of Na,CO, solution (20 mg of Na,CO,/lOO ml of isotonic saline). Convenient aliquots (0.3 ml) of the PG stock solutions were stored frozen for a maximum of 2 weeks. NE bitartrate (Nutritional Biochemical Co.), 5-HT creatinine sulfate complex (Sigma), and HIS dihydrochloride (Sigma) stock solutions were prepared in pH-adjusted (7.4) isotonic saline and kept frozen for no longer than 1 week. Identical vehicle solutions (without vasoactive agents) were prepared for controls. All stock solutions were thawed and further diluted with Ringer’s gelatin (1 .O%) solution immediately prior to use. Resultant concentrations of the vasoactive !agents were warmed (approx. 37.5”) and applied to the superior surface of the urinary bladder in 0. l-ml volumes without interruption of the Ringer’s super-fusion, according to the technique described by Messina et al. (1974).
PROSTAGLANDIN
EFFECTS
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Vessel diameter measurements were made with a stage-calibrated ocular micrometer in a Leitz biocular microscope at 100x. On the ventral aspect of the bladder, a microvascular bed with both 40- to 90-pm (outer vessel diameter) feeding arterioles (Zwiefach, 1968) and 50- to 125-pm (outer vessel diameter) muscular venules was selected for observation. Constrictions and dilatations were recorded as changes in vessel lumen diameter. Total elapsed time before initial response, maximum response, and return to control state were monitored. The microvascular bed was maintained in a control state for a minimum of 8 min between drug administrations. The vessel diameters were recorded immediately prior to and following the topical application of each vasoactive agent at varying dosage ranges which were determined by the degree of response. In each of six animals, each dose was tested for a vasoactive response on arterioles and venules simultaneously. The agents were tested in random order. However, within each agent dose range, test treatment order was from lowest to highest dosage. In an additional four animals, the effects of PGE, and HIS on arteriolar responsiveness to other vasoactive agents (NE, 5-HT, and PGF,,) were measured (Messina et uf., 1974). A single dose of a vasodilator (PGE,, 10 pg; or HIS, 100 pg) was given topically, to be followed in 10 min (the time interval which ensured return to control diameters) with the initial dose of a vasoconstrictor (PGF,, 100 kg; NE, 1 pg: or 5-HT, 1000 pg). The vasoconstrictors were then reapplied at lo-min intervals until the response was equivalent to the control values found in the previous six animals. Arteriolar lumen diameters were recorded prior to and following each application. The histochemical technique for localization of 15-hydroxy prostaglandin dehydrogenase (15OH PGDH), as originally reported by Nissen and Andersen (1968) and modified by Siggins (1972), was used to determine the primary site of PG deactivation in the urinary bladder. Rat urinary bladders were excised from four additional animals, minimally expanded with 0.5 ml of 4% gelatin solution, and immediately frozen in liquid nitrogen-cooled isopentane. Unfixed frozen sections were cut 8 pm thick on a cryostat (Ames Lab-Tek) at -20”. The cut sections were mounted on glass coverslips prior to incubation. A calibrated syringe was used to bathe each tissue section with 0.1 ml of incubation medium [diphosphopyridine nucleotide, 0.5 mg/ml; nitroblue tetrazolium, 0.5 mg/ml; and PGE, 0.5 mg/ml (from a stock solution of 1.0 mg of PGE,/O. 1 ml of ETOH/0.9 mg of Na,CO, solution)] in Tris buffer at a pH of 8.3. Two control incubation media were also prepared by eliminating (1) PGE, and (2) PGE, and absolute ethanol. The tissue sections were placed in an incubating oven at 37” for 60 min and then removed and mounted on slides with glycerin jelly for microscopic study. The data have been expressed as the mean changes from control measurements of arteriolar and venular diameters at each dose level for 10 vasoactive agents and the vehicle solution. Significance was determined with a two-tailed Student’s t test with an cr level of 0.05 (Kirk, 1968). -An analysis of variance for a randomized block design was used to detect significant differences in mean vessel diameter changes among dose levels for each agent and to determine significant differences among vasoactive agents at equal microgram doses (P = 0.05). Significant differences between vasoactive agents at equal dose levels were determined with the
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Newman-Kuels pairwise comparison procedure. A randomized block design and the Newman-Kuels pairwise comparison procedure were also utilized to determine significant differences between mean arteriole diameter changes among the three lo-min intervals (P = 0.05) (Kirk, 1968). RESULTS Vasomotor actions and relative’ potencies of NE, S-HT, HIS, and PGs El, Eu Flo,, F, A 1, and A*. Significant arteriolar dilatation occurred with PGE,, PGE2, PGA,, PGA,, PGF,, (all dose related), and HIS (not dose related), but not with the vehicle solution (Table 1). In general, PGE, and PGE, were more potent arteriolar dilators than PGA1, HIS, PGF,,, and PGA,. HIS, PGE,, PGE,, and PGA, also produced significant venodilation, for which HIS and PGE, were dose related. Significant dose-related arteriolar constriction occurred with NE, 5-HT, and PGF, (Table 2), whereas significant venoconstriction (dose related) was noted only with NE. The order of potency for arteriolar constrictors was NE > PGF, > 5-HT. Pretreatment effect of PGE, and HIS on the arteriolar responsiveness to PGF,, NE, and 5-HT. Arteriolar responses to PGF,, (100 pg) and NE (1 .O ,ug) at 10 and 20 min following PGE, (10 pg) were significantly less than the arteriolar constriction occurring at 30 min after PGE, (Table 3). Arteriolar constrictions with S-HT (1000 pg) at 10 and 20 min after PGE, were not statistically different and were comparable to the responses in the six animals with no pretreatment dilators. Arteriolar responses to PGF,, NE, and 5-HT following pretreatment with HIS (100 pg) were not significantly different between the lo- and 20-min intervals. There was significantly less arteriolar constriction with the PGE,-PGF,, series than with the HIS-PGF,, series at 10 and 20 min after the dilator. Also, the PGE,-NE series arteriolar constriction responses were less than the HIS-NE series, whereas the PGE,-5-HT series was not significantly different from the HIS-5-HT series. Histochemistry. The primary site of deactivation of PGE, was histochemically localized to bundles of smooth muscle fibers in the muscular coat of the rat urinary bladder wall. No 15-OH PGDH activity was noted in vascular wall or endothelium. DISCUSSION Vasomotor actions and relative potencies of NE, 5-HT, HIS, and PGs E,, EB Flu, F, A,andA,. The significant arteriolar dilatation following topical administration of PGs E,, Ez, A,, and A, is consistent with the indirect measurements (decreased total peripheral vascular resistance and a fall in systemic arterial pressure) following intravenous administration in rats (Holmes et al., 1963; Weeks and Wingerson, 1964; Horton and Main, 1963; Pike et al., 1967; Weeks et al., 1969). Additional direct evidence of the arteriolar dilatory activity of these PGs has been reported in several previous studies (Kaley and Weiner, 1968; Viguera and Sunahara, 1%9; Kaley et al., 1972; Siggins, 1972; Messina et al., 1974).
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PGE ,-PGF,
HWPGF, PGE,-NE HIS-NE
PGE,-5-HT HIS-5-HT
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Pretreatment agentvasoconstrictor pm
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pretreatmentO
NE, AND 5-HT
NS NS
NS’ 58.6 NS
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changes at equal doses of each vasoconstrictor (PGF,,, 100 fig: NE, 1.0 pg: 5-HT, 1000 pg) a vasodilator (PGE,, IO pg: HIS, 100 pg) were determined by an analysis of variance for a elapsed level was tested with the Newman-Keuls pairwise comparison procedure. and percentage) represents the mean decrease from control measurements in four animals.
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REPEATED MEASURES OF ARTERIOLAR VASOCONSTRICTOR RESPONSES TO PGF,,, FOLLOWING SINGLE PRETREATMENT WITH PGE, AND HIS”
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The application of PGF,, resulted in arteriolar dilatation and not arteriolar constriction as had been inferred from previous studies which used indirect measures to estimate the arteriolar vasomotor state (Pike ef al., 1967; Lavery et al., 1970). Other more direct studies have found the administration of PGF,, to result in no change in vascular resistence in the dog hindpaw (Kadowitz et al., 1971) and arteriolar dilatation in the hamster cheek pouch (Siggins, 1972). These differences may be explained by the presence or absence of PG receptors for vasoconstriction and vasodilation. Also, interactions of PG species with PG receptors may influence vascular responses. For example, PGF has been found to bind to PGE receptors (Kuehl et al., 1973). The arteriolar constriction observed with PGF,, is consistent with previous findings of topical administration of PGF,, (10 kg) on the feeding arterioles of the omentum uteri in the rat (Cseply and Csapo, 1972). Pretreatment effect of PGE, and HIS on the arteriolar responsiveness to PGF,,, NE, and 5-HT. The decreased arteriolar responsiveness to NE following PGE,, relative to NE following HIS, is consistent with similar previous findings in the dog (Hedwall et al., 1971; Kadowitz et al., 1971) and in the rat (Weiner and Kaley, 1969; Viguera and Sunahara, 1969; Messina et al., 1974). However, the time-related changes in arteriolar responsiveness to topical PGF,, following pretreatment with PGE, have not been previously reported. The time dependency for this interaction is probably secondary to redistribution of PGE, from its site of action. PGF,, has been shown to reverse the antagonistic effect of PGE, on the vasoconstrictor responses to nerve stimulation in the dog hindpaw (Kadowitz et al., 1971). The mechanisms resulting in such a PGF,,/PGE, interaction are not clear. Several studies support the proposed sites.of action of PGF,, and PGE, to be postjunctional and post-p receptor, respectively. The vasodilatory activity of PGE, is not diminished by atropine (Tiirker et al., 1968)or p blockade (Daugherty, 1971; Ulano et al., 1972), but is associated with increased concentrations of adenosine 3’, Y-monophosphate (cyclic AMP) (Shepherd et al., 1973). Similarly, the vasoconstrictor activity of PGFza is not affected by ganglionic blockade (Mark et al., 1971), but is associated with increased guanosine 3’, 5’-monophosphate (cyclic GMP) levels (Kadowitz et al., 1975), increased cyclic GMP/cyclic AMP ratios (Dunham et al., 1974), and increased free Ca”+ levels (Carsten, 1972). Thus, possible sites of the inhibitory effect of PGE, on the arteriolar responsiveness to PGF,, may be: (1) inhibition at the PGF receptor site; (2) indirect antagonism at the cyclic nucleotide level, with the resultant cyclic GMP/cyclic AMP ratio being the primary determinant of vasoactivity; or (3) an unknown locus between the (Y receptor and eventual release of Ca2+ into the myoplasm. Histochemistry. Histochemical localization of 15OH PGDH in urinary bladder wall smooth muscle and not in vascular smooth muscle or endothelium may reflect the bladder vasculature’s lack of or reduced capability for metabolism of PGs in blood. It has been well documented that lung tissue is capable of removing and metabolizing PGs from the pulmonary circulation (Ferreira and Vane, 1967; Piper et al., 1970; Anderson and Eling, 1976). It is yet to be determined if PGs, circulating in urinary bladder blood vessels, pass unchanged through that vascular
PROSTAGLANDIN
EFFECTS
IN MICROCIRCULATION
9
bed for subsequent removal and metabolism. Histochemical evidence for 15OH PGDH in smooth muscle of the urinary bladder wall supports the suggestion by Piper and Vane (1971) that local control by PCs and their metabolic enzymes may allow storage organs such as stomach or bladder to accommodate to stretch and thus minimize cell distortion. Such a mechanism in urinary bladder may involve released PC as smooth muscle relaxants and PC metabolic enzymes as modulators which help ensure that tension in smooth muscle of the wall does not increase proportionally to filling. SUMMARY
AND CONCLUSION
In most cases, topical applications of PCs resulted in dose-related arteriolar diameter changes. PGE, and PGE, were the most potent vasodilators; PGF,, was the most potent arteriolar constrictor. PGF,, produced arteriolar dilatation in the rat urinary bladder. Evidence was found for an apparent inhibitory effect of PGE, on the actions of PGF,, and NE, implying that PCs play an integral role in local blood flow modulation to support the changing metabolic needs of the urinary bladder wall. 15OH PGDH histochemical activity being localized in bladder smooth muscle, and not in vascular smooth muscle or endothelium, infers that circulating PCs pass to an organ such as lung for their metabolism and that degradative enzyme activity in the urinary bladder wall modulates local effects of endogenous PCs. ACKNOWLEDGMENT The authors are grateful to Dr. J. E. Pike of the Upjohn Company, Kalamazoo, Michigan, for the supply of prostaglandins.
REFERENCES ANDERSON, M. W., AND ELING, T. E. (1976). Prostaglandin removal and metabolism by isolated perfused lung. Prostaglundins 2, 645-677. CARSTEN,M. E. (1972). Prostaglandin’s part in regulating uterine contraction by transport of Ca++. In “The Prostaglandins: Clinical Application in Human Reproduction” (E. M. Southern, ed.), pp. 59-66. Futura, Mt. Kisco, New York. CSEPLY, J., AND CSARO,A. I. (1972). The effect of prostaglandin F,, on the small arteries of the omentum uteri in the rat. Prostaglandins 1, 235-238. DAUGHERTY,R. M., JR. (1971). Effects of iv and ia prostaglandin E, on dog forelimb skin and muscle blood flow. Amer. J. Physiol. 220, 392-396. DUCHARME, D. W., AND WEEKS,J. R. (1967). Cardiovascular pharmacology of prostaglandin F,, a unique pressor agent. In “Nobel Symposium 2, Prostaglandins” (Cl. Bergstrom and B. Samuelsson, eds.), pp. 173-182. Interscience, New York. DUNHAM, E. W., HADDOX, M. K., AND GOLDBERG,N. D. (1974). Alteration of vein cyclic 3’5 nucleotide concentrations during changes in contractility. Proc. Nut. Acad. Sci. USA 71, 815-819. ECHT, R., DEY, R. D., AND YOUNG, W. F., JR. (1976). Internal tissue heat during in viva transillumination with fused quartz and borosilicate glass rods. Microvasc. Res. 11, 217-226. FERREIRA,S. H., AND VANE, J. R. (1967). Prostaglandins: Their disappearance from and release into the circulation. Nature (London) 216, 868-873. HEDWALL, P. R., ABDEL-SAYED, W. A., SCHMID, P. G., MARK, A. L., AND ABBOUD, F. M. (1971). Vascular responses to prostaglandin E, in gracilis muscle and hindpaw of the dog. Amer. J. Ph.vsiol. 221. 42-47.
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HOLMES, S. W., HORTON, E. W., AND MAIN, I. H. M. (1963). The effect of prostaglandin E, on responses of smooth muscle to catecholamines, angiotensin and vasopressin. Byit. J. Pharmacol. 21, 538-543.
HORTON, E. W., AND MAIN, I. H. M. (1963). A comparison of biological activities of four prostaglandins. Brit. J. Pharmacol. 21, 182-189. KADOWITZ, P. J., JOINER, P. D., HYMAN, A. L., AND GEORGE,W. J. (1975). Influence of prostaglandin E, and F,, on pulmonary vascular resistence, isolated lobar vessels and cyclic nucleotide levels. .I. Pharmacol.
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KADOWITZ, P. J., SWEET, C. S., AND BRODY, M. J. (1971). Differential effects of prostaglandins E,, E,, F,, and F,, on adrenergic vasoconstriction in the dog hindpaw. J. Pharmacol. Exp. Ther. 177, 641-649.
KALEY, G., MESSINA, E. J., AND WEINER, R. (1972). The role of prostaglandins in microcirculatory regulation and inflammation. In “Prostaglandins in Cellular Biology” (P. W. Ramwell and B. B. Pharriss, eds.), pp. 309-327. Plenum Press, New York-London. KALEY, G., AND WEINER, R. (1968). Microcirculatory studies with prostaglandin E,. In “Prostaglandin Symposium of the Worchester Foundation for Experimental Biology” (P. W. Ramwell and J. E. Shaw, eds.), pp. 321-328. Interscience, New York. KIRK, R. (1968). “Experimental Design.” Brooks/Cole, Belmont, Calif. KNISELY, M. H. (1936). A method of illuminating living structures for microscopic study. Anaf. Rec. 64, 449-524.
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LAVERY, H. A., LOWE, R. D., AND SCROOP,G. C. (1970). Cardiovascular effects of prostaglandins mediated by the central nervous system of the dog. Brir. J. Pharmacol. 39, 511-519. MARK, A. L., SCHMID,~P.G., ECKSTEIN,J. W., AND WENDLING, M. G. (1971). Venous responses to prostaglandin F,,. Amer. J. Physiol. 220, 222-236. MESSINA, E. J., WEINER, R., AND KALEY, G. (1974). Microcirculatory effects of prostaglandins E,, E,, and A, in the rat mesentery and cremaster muscle. Microvasc. Res. 8, 77-89. NISSEN, H. M., AND ANDERSEN, H. (1968). On the localization of prostaglandin-dehydrogenase activity in the kidney. Histochemie 14, 189-200. PIKE, J. E., KUPIECKI, F. P., AND WEEKS, J. P. (1967). Biological activity of the prostaglandins and related analogs. In “Nobel Symposium 2, Prostaglandins” (G. Vergstrom and B. Samuelsson, eds.), pp. 161-171. Interscience, New York. PIPER, P. J., AND VANE, J. R. (1971). The release of prostaglandins from lung and other tissues. Ann. N. Y. Acad. Sci. 180, 363-385.
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PITTS, R. F. (1968). “Physiology of the Kidney and Body Fluids,” pp. 227-228. Yearbook Medical, Chicago. SHEPHERD,A. P., MAO, C. C., JACOBSON,E. D., AND SHANBOUR,L. L. (1973). The role of cyclic AMP in mesenteric vasodilation. Microvasc. Res. 6, 332-341. SIGGINS,G. R. (1972). Prostaglandins and the microvascular system: Physiological and histochemical correlations. In “Prostaglandins in Cellular Biology and the Inflammatory Process” (P. W. Ramwell and B. B. Pharris, eds.), pp. 451-478. Plenum Press, New York. TORKER, R. K., KAYMAKCALAN, S., AND AYHAN, I. H. (1968). Effect of prostaglandin E, on vascular responsiveness to norepinephrine and angiotensin in the anesthetized rat. Arzneimirrel.-Forschung 18, 1310-1312.
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RESEARCH
17, I-11 (1979)
Comparisons of Prostaglandin Vasoactive Effects and Interactions in the in Viva Microcirculation of the Rat Urinary Bladder’ WILLIAM
F. YOUNG,JR.,
RICHARD D. DEY, AND ROBERT ECHT
Department of Anatomy, Michigan State University, Received
College of Human Medicine, East Lansing, Michigan 48824 May 26, 1978
A combination of techniques for in vivo transillumination, topical application of vasoactive agents, and direct microscopic observation of microcirculatory responses was utilized to evaluate the vasomotor actions of prostaglandins (PGs) E,, E,, F,,, F,, A,, and A, on rat urinary bladder arterioles and venules. The effects of PGE, and histamine (HIS) on arteriolar responsiveness to norepinephrine (NE), serotonin (5HT), and PGF, were measured. Histochemical studies were completed to determine the primary site of prostaglandin (PG) metabolic deactivation in the urinary bladder. Arteriolar dilatation occurred with HIS, PGE,, PGE,, PGA,, PGAf, and PGF,,, all of which (with the exception of HIS) demonstrated significant dose-related responses. Overall, PGE, and PGEZ were of greatest potency. Significant dose-related arteriolar constriction occurred with NE > PGF, > 5-HT (in order of decreasing potency). HIS, PGE,, PGE,, and PGA, produced significant venular dilatation; PGE, and HIS were dose related. Only NE resulted in significant venoconstriction. Arteriolar responsiveness to NE and PGF,, decreased after pretreatment with PGE, but was unchanged by HIS pretreatment, whereas application of 5-HT following pretreatment with PGE, or HIS produced equivalent levels of arteriolar constriction. The primary site of deactivation of PGE, was histochemically localized to bundles of smooth muscle fibers in the muscular coat of the rat urinary bladder wall.
INTRODUCTION Several previous investigations have provided models for defining the in viva vascular responses to exogenous prostaglandins (PGs).~ The most direct in vivo method of experimentation consists of local applications of the PGs and direct observations of their effects on the microcirculation (Kaley and Weiner, 1968; Weiner and Kaley, 1969; Siggins, 1972; Messinaer af., 1974). In this investigation, a combination of techniques for in viva transillumination, topical applications of vasoactive agents, and direct microscopic observations of the rat urinary bladder microvasculature was utilized to evaluate: (1) the vasomotor actions and relative potencies of norepinephrine (NE), serotonin (5-HT), histamine (HIS), and PGs El, E,, F1,, FZu, A,, and A,; and (2) the pretreatment effect of PGE, and HIS on the arteriolar responsiveness to PGF,,, NE, and 5-HT. In addition, histochemical * This study was supported in part by a grant from the Michigan Heart Association. Send reprint requests to Dr. Robert Echt, Department of Anatomy, College of Human Medicine, Michigan State University, East Lansing, Michigan 48824. L Abbreviations used: PGs, prostaglandins; PG, prostaglandin; NE, norepinephrine; 5-HT, serotonin, 5-hydroxytryptamine; HIS, histamine; 15-OH PGDH, 15-hydroxy prostaglandin.dehydrogenase; cyclic AMP, adenosine 3’, 5’-monophosphate; cyclic GMP, guanosine 3’, 5’-monophosphate. 1 00262862/79/01O~WCt-11$02.00/0 Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.
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studies were also completed to determine the primary site of PG metabolic deactivation in the rat urinary bladder. MATERIALS
AND METHODS
Anesthesia of male and female mature rats (200-325 g) was induced with an intraperitoneal injection of sodium pentobarbital (Jen-Sal), 30-50 mg/kg, and maintained with supplementary subcutaneous doses of 6 mg/kg every 20 min. A tracheotomy permitted adequate ventilatory exchange and decreased the body movement secondary to respiratory efforts. A ventral, abdominal, midline incision was made superior to the urinary bladder, which was then exposed with the peritoneum intact. The ureters were severed to ensure static bladder size and intraviscus pressure. The inferior and superior surfaces of the urinary bladder were continuously superfused with warmed (37.2”) mammalian Ringer’s solution flowing at a constant rate of 2 mllmin. The pH of the Ringer’s solution was adjusted to 7.4 with dry sodium bicarbonate. In four control animals, an indwelling bladder cannula was connected to a low-pressure transducer (Statham P23BB) to cystometrically monitor intrabladder pressures (Pitts, 1968). As warmed Ringer’s solution was infused in OS-ml increments, stable internal bladder pressures (11.5-14.0 mm Hg) were recorded between bladder volumes of 0.5 and 1.5 ml. Transverse and longitudinal bladder diameters were then measured with a caliper and correlated with bladder volumes. In all subsequent animals, repeated diameter measurements were made to ensure bladder volumes within the standardized range for consistent internal pressures. The apparatus first described by Knisely (1936) and recently modified by Wilson (1970) and Echt et al. (1976) was utilized for in vivo transillumination of the urinary bladder. Copper-constantan 25-gauge thermocouples were used for continuous monitoring of superior dripper, borosilicate glass rod tip (source of illumination and inferior super-fusion), and rectal temperatures. Urinary bladder surface temperatures were intermittently monitored with 40-gauge thermocouples. Body temperature was maintained in a cylindrical heating chamber made from tin and wrapped with electrical tape (Smith-Gates Corp.). All temperatures were recorded on a Grass polygraph and maintained at 37.2 + 0.5”. PG (Upjohn) stock solutions were prepared by dissolving 1.OOmg of each PG in 0.1 ml of absolute ethanol and then adding 0.9 ml of Na,CO, solution (20 mg of Na,CO,/lOO ml of isotonic saline). Convenient aliquots (0.3 ml) of the PG stock solutions were stored frozen for a maximum of 2 weeks. NE bitartrate (Nutritional Biochemical Co.), 5-HT creatinine sulfate complex (Sigma), and HIS dihydrochloride (Sigma) stock solutions were prepared in pH-adjusted (7.4) isotonic saline and kept frozen for no longer than 1 week. Identical vehicle solutions (without vasoactive agents) were prepared for controls. All stock solutions were thawed and further diluted with Ringer’s gelatin (1 .O%) solution immediately prior to use. Resultant concentrations of the vasoactive !agents were warmed (approx. 37.5”) and applied to the superior surface of the urinary bladder in 0. l-ml volumes without interruption of the Ringer’s super-fusion, according to the technique described by Messina et al. (1974).
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Vessel diameter measurements were made with a stage-calibrated ocular micrometer in a Leitz biocular microscope at 100x. On the ventral aspect of the bladder, a microvascular bed with both 40- to 90-pm (outer vessel diameter) feeding arterioles (Zwiefach, 1968) and 50- to 125-pm (outer vessel diameter) muscular venules was selected for observation. Constrictions and dilatations were recorded as changes in vessel lumen diameter. Total elapsed time before initial response, maximum response, and return to control state were monitored. The microvascular bed was maintained in a control state for a minimum of 8 min between drug administrations. The vessel diameters were recorded immediately prior to and following the topical application of each vasoactive agent at varying dosage ranges which were determined by the degree of response. In each of six animals, each dose was tested for a vasoactive response on arterioles and venules simultaneously. The agents were tested in random order. However, within each agent dose range, test treatment order was from lowest to highest dosage. In an additional four animals, the effects of PGE, and HIS on arteriolar responsiveness to other vasoactive agents (NE, 5-HT, and PGF,,) were measured (Messina et uf., 1974). A single dose of a vasodilator (PGE,, 10 pg; or HIS, 100 pg) was given topically, to be followed in 10 min (the time interval which ensured return to control diameters) with the initial dose of a vasoconstrictor (PGF,, 100 kg; NE, 1 pg: or 5-HT, 1000 pg). The vasoconstrictors were then reapplied at lo-min intervals until the response was equivalent to the control values found in the previous six animals. Arteriolar lumen diameters were recorded prior to and following each application. The histochemical technique for localization of 15-hydroxy prostaglandin dehydrogenase (15OH PGDH), as originally reported by Nissen and Andersen (1968) and modified by Siggins (1972), was used to determine the primary site of PG deactivation in the urinary bladder. Rat urinary bladders were excised from four additional animals, minimally expanded with 0.5 ml of 4% gelatin solution, and immediately frozen in liquid nitrogen-cooled isopentane. Unfixed frozen sections were cut 8 pm thick on a cryostat (Ames Lab-Tek) at -20”. The cut sections were mounted on glass coverslips prior to incubation. A calibrated syringe was used to bathe each tissue section with 0.1 ml of incubation medium [diphosphopyridine nucleotide, 0.5 mg/ml; nitroblue tetrazolium, 0.5 mg/ml; and PGE, 0.5 mg/ml (from a stock solution of 1.0 mg of PGE,/O. 1 ml of ETOH/0.9 mg of Na,CO, solution)] in Tris buffer at a pH of 8.3. Two control incubation media were also prepared by eliminating (1) PGE, and (2) PGE, and absolute ethanol. The tissue sections were placed in an incubating oven at 37” for 60 min and then removed and mounted on slides with glycerin jelly for microscopic study. The data have been expressed as the mean changes from control measurements of arteriolar and venular diameters at each dose level for 10 vasoactive agents and the vehicle solution. Significance was determined with a two-tailed Student’s t test with an cr level of 0.05 (Kirk, 1968). -An analysis of variance for a randomized block design was used to detect significant differences in mean vessel diameter changes among dose levels for each agent and to determine significant differences among vasoactive agents at equal microgram doses (P = 0.05). Significant differences between vasoactive agents at equal dose levels were determined with the
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Newman-Kuels pairwise comparison procedure. A randomized block design and the Newman-Kuels pairwise comparison procedure were also utilized to determine significant differences between mean arteriole diameter changes among the three lo-min intervals (P = 0.05) (Kirk, 1968). RESULTS Vasomotor actions and relative’ potencies of NE, S-HT, HIS, and PGs El, Eu Flo,, F, A 1, and A*. Significant arteriolar dilatation occurred with PGE,, PGE2, PGA,, PGA,, PGF,, (all dose related), and HIS (not dose related), but not with the vehicle solution (Table 1). In general, PGE, and PGE, were more potent arteriolar dilators than PGA1, HIS, PGF,,, and PGA,. HIS, PGE,, PGE,, and PGA, also produced significant venodilation, for which HIS and PGE, were dose related. Significant dose-related arteriolar constriction occurred with NE, 5-HT, and PGF, (Table 2), whereas significant venoconstriction (dose related) was noted only with NE. The order of potency for arteriolar constrictors was NE > PGF, > 5-HT. Pretreatment effect of PGE, and HIS on the arteriolar responsiveness to PGF,, NE, and 5-HT. Arteriolar responses to PGF,, (100 pg) and NE (1 .O ,ug) at 10 and 20 min following PGE, (10 pg) were significantly less than the arteriolar constriction occurring at 30 min after PGE, (Table 3). Arteriolar constrictions with S-HT (1000 pg) at 10 and 20 min after PGE, were not statistically different and were comparable to the responses in the six animals with no pretreatment dilators. Arteriolar responses to PGF,, NE, and 5-HT following pretreatment with HIS (100 pg) were not significantly different between the lo- and 20-min intervals. There was significantly less arteriolar constriction with the PGE,-PGF,, series than with the HIS-PGF,, series at 10 and 20 min after the dilator. Also, the PGE,-NE series arteriolar constriction responses were less than the HIS-NE series, whereas the PGE,-5-HT series was not significantly different from the HIS-5-HT series. Histochemistry. The primary site of deactivation of PGE, was histochemically localized to bundles of smooth muscle fibers in the muscular coat of the rat urinary bladder wall. No 15-OH PGDH activity was noted in vascular wall or endothelium. DISCUSSION Vasomotor actions and relative potencies of NE, 5-HT, HIS, and PGs E,, EB Flu, F, A,andA,. The significant arteriolar dilatation following topical administration of PGs E,, Ez, A,, and A, is consistent with the indirect measurements (decreased total peripheral vascular resistance and a fall in systemic arterial pressure) following intravenous administration in rats (Holmes et al., 1963; Weeks and Wingerson, 1964; Horton and Main, 1963; Pike et al., 1967; Weeks et al., 1969). Additional direct evidence of the arteriolar dilatory activity of these PGs has been reported in several previous studies (Kaley and Weiner, 1968; Viguera and Sunahara, 1%9; Kaley et al., 1972; Siggins, 1972; Messina et al., 1974).
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HWPGF, PGE,-NE HIS-NE
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changes at equal doses of each vasoconstrictor (PGF,,, 100 fig: NE, 1.0 pg: 5-HT, 1000 pg) a vasodilator (PGE,, IO pg: HIS, 100 pg) were determined by an analysis of variance for a elapsed level was tested with the Newman-Keuls pairwise comparison procedure. and percentage) represents the mean decrease from control measurements in four animals.
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The application of PGF,, resulted in arteriolar dilatation and not arteriolar constriction as had been inferred from previous studies which used indirect measures to estimate the arteriolar vasomotor state (Pike ef al., 1967; Lavery et al., 1970). Other more direct studies have found the administration of PGF,, to result in no change in vascular resistence in the dog hindpaw (Kadowitz et al., 1971) and arteriolar dilatation in the hamster cheek pouch (Siggins, 1972). These differences may be explained by the presence or absence of PG receptors for vasoconstriction and vasodilation. Also, interactions of PG species with PG receptors may influence vascular responses. For example, PGF has been found to bind to PGE receptors (Kuehl et al., 1973). The arteriolar constriction observed with PGF,, is consistent with previous findings of topical administration of PGF,, (10 kg) on the feeding arterioles of the omentum uteri in the rat (Cseply and Csapo, 1972). Pretreatment effect of PGE, and HIS on the arteriolar responsiveness to PGF,,, NE, and 5-HT. The decreased arteriolar responsiveness to NE following PGE,, relative to NE following HIS, is consistent with similar previous findings in the dog (Hedwall et al., 1971; Kadowitz et al., 1971) and in the rat (Weiner and Kaley, 1969; Viguera and Sunahara, 1969; Messina et al., 1974). However, the time-related changes in arteriolar responsiveness to topical PGF,, following pretreatment with PGE, have not been previously reported. The time dependency for this interaction is probably secondary to redistribution of PGE, from its site of action. PGF,, has been shown to reverse the antagonistic effect of PGE, on the vasoconstrictor responses to nerve stimulation in the dog hindpaw (Kadowitz et al., 1971). The mechanisms resulting in such a PGF,,/PGE, interaction are not clear. Several studies support the proposed sites.of action of PGF,, and PGE, to be postjunctional and post-p receptor, respectively. The vasodilatory activity of PGE, is not diminished by atropine (Tiirker et al., 1968)or p blockade (Daugherty, 1971; Ulano et al., 1972), but is associated with increased concentrations of adenosine 3’, Y-monophosphate (cyclic AMP) (Shepherd et al., 1973). Similarly, the vasoconstrictor activity of PGFza is not affected by ganglionic blockade (Mark et al., 1971), but is associated with increased guanosine 3’, 5’-monophosphate (cyclic GMP) levels (Kadowitz et al., 1975), increased cyclic GMP/cyclic AMP ratios (Dunham et al., 1974), and increased free Ca”+ levels (Carsten, 1972). Thus, possible sites of the inhibitory effect of PGE, on the arteriolar responsiveness to PGF,, may be: (1) inhibition at the PGF receptor site; (2) indirect antagonism at the cyclic nucleotide level, with the resultant cyclic GMP/cyclic AMP ratio being the primary determinant of vasoactivity; or (3) an unknown locus between the (Y receptor and eventual release of Ca2+ into the myoplasm. Histochemistry. Histochemical localization of 15OH PGDH in urinary bladder wall smooth muscle and not in vascular smooth muscle or endothelium may reflect the bladder vasculature’s lack of or reduced capability for metabolism of PGs in blood. It has been well documented that lung tissue is capable of removing and metabolizing PGs from the pulmonary circulation (Ferreira and Vane, 1967; Piper et al., 1970; Anderson and Eling, 1976). It is yet to be determined if PGs, circulating in urinary bladder blood vessels, pass unchanged through that vascular
PROSTAGLANDIN
EFFECTS
IN MICROCIRCULATION
9
bed for subsequent removal and metabolism. Histochemical evidence for 15OH PGDH in smooth muscle of the urinary bladder wall supports the suggestion by Piper and Vane (1971) that local control by PCs and their metabolic enzymes may allow storage organs such as stomach or bladder to accommodate to stretch and thus minimize cell distortion. Such a mechanism in urinary bladder may involve released PC as smooth muscle relaxants and PC metabolic enzymes as modulators which help ensure that tension in smooth muscle of the wall does not increase proportionally to filling. SUMMARY
AND CONCLUSION
In most cases, topical applications of PCs resulted in dose-related arteriolar diameter changes. PGE, and PGE, were the most potent vasodilators; PGF,, was the most potent arteriolar constrictor. PGF,, produced arteriolar dilatation in the rat urinary bladder. Evidence was found for an apparent inhibitory effect of PGE, on the actions of PGF,, and NE, implying that PCs play an integral role in local blood flow modulation to support the changing metabolic needs of the urinary bladder wall. 15OH PGDH histochemical activity being localized in bladder smooth muscle, and not in vascular smooth muscle or endothelium, infers that circulating PCs pass to an organ such as lung for their metabolism and that degradative enzyme activity in the urinary bladder wall modulates local effects of endogenous PCs. ACKNOWLEDGMENT The authors are grateful to Dr. J. E. Pike of the Upjohn Company, Kalamazoo, Michigan, for the supply of prostaglandins.
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