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Hanseatic endocrine conference on tissue prorenin-renin-angiotensin systems
ELSEVIER
MEETING REPORT Hanseatic Endocrine Conference on Tissue Prorenin-Renin-Angiotensin Systems LocalRegulatory Actions in Reproductive and Endocrine Organs, Hamburg, July H-16,1994 Amal K. Mukhopadhyay
Over the past decade, substantial evidence has become available implicating tissue prorenin-renin-angiotensin systems (PRAS) in the local regulation of differentiated cell functions within the tissues where it is expressed. In contrast to a mixed enzyme-endocrine role classically ascribed to the renal renin-angiotensin system in the context of cardiovascular homeostasis, the tissue PRAS appears to play an autocrine, paracrine, or even an intracrine regulatory role. It has been well documented that PRAS is expressed in a wide variety of tissues, including reproductive and endocrine glands. We are now poised to take major strides toward understanding such fundamental issues as, for example, the tissue-specific regulation of the expression of PRAS, the nature of signal transduction pathways associated with various angiotensin II (AII) receptors, and the relevance of an aberrant expression of the tissue PRAS to pathophysiology encountered in a number of endocrine and reproductive organs. This symposium was organized as a satellite to the Third European Endocrinology Congress, Amsterdam, The Netherlands, to focus the discussion on current advances in these important aspects of tissue PRAS and to generate (Trends Endocrinol Metab 1995;6:8ideas on future research strategies. 10). The symposium started with a discussion on molecular aspects of the reninangiotensin system, including the regulation of tissue-specific expression of renin gene and the application of transgenic strategy. M. Paul (Berlin, Germany) briefly reviewed the available
data on gene expression of various components of prorenin-renin-angiotensin systems (PRAS) in several extrarenal organs. The link between the aberrant gene regulation of angiotensin-converting enzyme (ACE) and various cardiovascular abnormalities such as hypertension, myocardial infarction, and cardiac hypertrophy was particularly stressed by him. J. Mullins (Edinburgh, Amai K. Mukhopadhyayis at the Institutefor UK) discussed the benefits of transgenic Hormone and Fertility Research, University of Hamburg, Grandweg 64, D-22529 Ham- strategies to the understanding of the role of extrarenal PRAS. Transgenic rats burg, Germany. 8
01995,
EIsevIer Science Inc., 1043-2760/95/$9.50
SSDI
were produced by introducing a fusion transgene where a cDNA sequence encoding ren-2 was driven by a,-antitrypsin promoter. Such animals were characterized by extreme cardiac hypertrophy leading to an early death of the transgenic founders. Data from one that survived showed that the cardiac hypertrophy observed in these animals could be directly linked to the high level of extrarenal prorenin in their circulation, resulting from the expression of yen-2 gene solely in the liver. This once again underlined the importance of extrarenal prorenin with reference to the development of cardiovascular pathology. T. Voigtlander (Berlin, Germany) reported that intron I of the rat renin gene possibly contains a silencer involved in its tissue-specific expression. Such information on the transcriptional regulation of the renin gene are valuable for the understanding of both its normal and aberrant expression in extrarenal tissues. One of the complications of cardiovascular diseases associated with an increased circulatory renin level is the hypertrophy observed in cardiovascular and renal tissues, as just described. AI1 acts as a hypertrophic factor for smooth muscle cells in these tissues (W.A. Hsueh, Los Angeles, CA, USA; and G. Wolf, Hamburg, Germany). In cardiac tissues, according to W.A. Hsueh, AI1 acts on cardiac fibroblasts, which are richly endowed with AT, receptors to promote collagen and extracellular matrix production. Additionally, growth factors produced by such activated fibroblasts are responsible for the growth effects on cardiac myocytes in a paracrine manner. G. Wolf proposed that, in proximal tubular cells of the kidney, AII-induced cellular hypertrophy was mediated by transforming growth factor B (TGF@, since AI1 increased both the rate of transcription and the synthesis of TGFB in these cells and a neutralizing antiserum to TGFl3 could block the growth effects of AII. In cells like mesangial cells, however, where AI1 is mitogenic,
1043-2760(94)00091-H
TEM Vol. 6,No.1,1995
no induction of TGF@ was Thus, whether AI1 stimulation a cellular depends
hypertrophy on whether
observed. results in
or proliferation TGFfl is expressed
or not, which in turn is determined
by
thus appear to persist regarding the possible signal transduction cascades utilized by AT,. This remains, therefore, an
interesting
exploration.
area
requiring
Also, questions
further
such
as if
the ability of AI1 to induce certain trans-acting factors in a cell-specific manner. In the last talk of this session, using an interesting model of in-fieldstimulated guinea-pig atria, H. Brasch (Ltibeck, Germany) clearly showed that AI1 could stimulate the release of norepinephrine from sympathetic nerve endings by an activation of AT, receptors, and postulated that in this process AT, receptors somehow interact with c+ adrenoceptors. Knowledge of the molecular biology, physiology, and pharmacology of angiotensin receptors is fundamental to an understanding of how AI1 is able to bring about its diverse biologic effects in a variety of tissues. This was the theme of the third session of the first day, where interesting, but at times somewhat divergent, views were presented by S. Bottari (Grenoble, France) and T. Inagami (Nashville, TN, USA). According to the former, AI1 binds to two pharmacologically distinct receptor subtypes, AT, and AT,. Based on molecular cloning data, Inagami was able to subdivide the AT, type into subtypes AT,,, predominantly expressed in adrenal and pituitary, and which has 96% homology with AT,,, AT,, and is abundantly expressed in contractile and renal cells. Both subtypes belong to the seven transmembrane domain type receptor class acting via Gi or G, and being linked to diverse signaling pathways, including an inhibition of adenylate cyclase, activation of phospholipase A, C, or D, and the regulation of calcium channels. According to Bottari, AT, receptors, when activated in a ligand-specific manner, stimulate a phosphotyrosinephosphatase activity via a G-protein-independent mechanism. Such activation of phosphotyrosine phosphatase is, in an as yet unknown manner, linked to an inhibition of ANPreceptor-coupled guanylate cyclase activity. Expression cloning of the AT,
other classes or subclasses of AI1 receptors exist and if yes, where these are localized (at the cell surface or within the cell) will continue to engage scientific attention in the future. In this context, G. Vinson (London, UK), using a monoclonal antibody specifically directed against the N-terminal region of AT, receptors, showed that AT, receptors on adrenal
receptor yielded data which, according to Inagami, convincingly demonstrated that this receptor contains seven transmembrane domains and is linked to an activation of certain specific phosphotyrosine phosphatase, in a G-proteindependent manner. Some uncertainties
neuronal cell culture from the brain of SHR rats had not only more AT, receptor, but also a greater amount of the corresponding mRNA. Such high expression of AT, receptors in the brain of SHR rats may be responsible for a hyperactive renin-angiotensin system in the brain of
TEA4Vol. 6,No.1,1995
glomerulosa cells continuously cycle between the plasma membrane and intracellular locations. Stimulation of steroidogenesis by AII, however, does not require internalization of the receptors, as in cells where receptors were anchored to plasma membrane by the antibody, AI1 could still stimulate aldosterone production. J. Peters (Heidelberg, Germany), using the immunogold labeling technique, showed ultrastructural data that renin and ACE activities are localized within the rat adrenal mitochondria. Also, AII-binding sites were demonstrated on membrane preparations from purified mitochondria. Thus, a local role of AI1 at the mitochondrial level in regulating adrenal steroidogenesis was postulated. Such intracrine regulatory pathways are increasingly discussed with respect to tissue PRAS. Obviously, further studies are necessary to explore this interesting possibility. The next session was devoted to a discussion of the role of angiotensin as a neuroendocrine regulator, especially in the central nervous system. Tissue PRAS in the brain has been investigated by many laboratories to elucidate its role in the development of hypertension. Compared with normotensive WKY rats, SHR rats are known to possess a hyperactive renin-angiotensin system. Working on this model, M.K. Raizada (Gainesville, FL, USA) reported that the numbers of AT, receptors in neuronal cell cultures from the brain of SHR rats were vastly increased. Compared with WKY rats, the
01995, Elsevier Science Inc., 1043-2760/95/$9.50
SSDI
these animals. On the other hand, D.J. Campbell (Heidelberg, Australia) raised questions about the relative importance of brain AI1 in developing high blood pressure in SHR rats, since he could measure only extremely low levels of AI1 in the brain. The importance of brain angiotensin peptides in regulating the secretion of pituitary trophic hormones has been known for a long time. M.I. Phillips (Gainesville, FL, USA) described experiments designed to test the hypothesis that AI1 in the hypothalamus may be involved in the preovulatory luteinizing hormone (LH) surge. Using a model of ovariectomized estrogen-progesteroneprimed rats, it was shown that the LH surge in these animals was indeed preceded by a peak of hypothalamic AI1 level. This led Phillips to conclude that AI1 neurons in the hypothalamus may be implicated in the regulation of the LH surge. AI1 may not only perform in the regulation of trophic hormone release from the pituitary but may also influence the growth of the pituitary, since AI1 is known to be able to induce hyperplasia or cell proliferation in a number of tissues (see earlier discussion here). This question was addressed by M. Pawlikowski (Lodz, Poland). AI1 at subnanomolar concentrations stimulated radiolabeled thymidine incorporation in rat pituitary tumor cells. The cell proliferation indices were diminished by ACE inhibitors and the AI1 receptor antagonists losartan and PD123177. Three sessions on the second day were earmarked for discussions on the role of the renin-angiotensin system within reproductive tissues, such as ovary, testis, and the uteroplacental unit. F. Naftolin (New Haven, CT, USA) opened the second day with an in-depth review of the available evidence on the intraovarian regulatory role of PRAS. He underscored the role of AI1 produced within the ovary as an important regulator of ovulation. In addition, he highlighted the role of other bioactive angiotensin peptides like angiotensin III, angiotensin 1-7, and angiotensin 3-8. Clearly, more studies are necessary to define the intraovarian role of these peptides. Association of ovarian PRAS with follicular atresia in rat was convincingly demonstrated by A. Husain (Cleveland, OH, USA). Along with the expression of AT, receptors, the atretic granulosa cells start expressing
1043-2760(94)00091-H
9
ACE activity and the level of follicular fluid renin rises. He noted that ACE, but not AT, receptors, could be found also on nonatretic granulosa cells. A possible relevance of PRAS to male reproduction was discussed by A. Mukhopadhyay (Hamburg, Germany), who demonstrated the presence of prorenin in sperm-free human seminal fluid samples by a direct immunoradiometric assay, which did not require any prior enzymatic activation of prorenin. Western blotting procedure identified a 48-kD protein as prorenin in the seminal fluid, and there was a direct correlation between sperm density and the level of prorenin in the seminal fluid. The origin of prorenin in semen remains obscure, and major research efforts will be needed to discern if seminal PRAS is relevant for sperm function. Possible involvement of ovarian PRAS in the etiology of the ovarian hyperstimulation syndrome was discussed by R.S. Morris (Los Angeles, CA, USA), who proposed that the use of ACE inhibitors may ameliorate the severity of the disease by reducing vascular permeability. That the PRAS could be relevant to polycystic ovarian disease (PCOD) was suggested by I. Matinlauri (Turku, Finland), who observed a significantly greater elevation of total renin in circulation after gonadotropin stimulation in PCOD patients compared with a control group consisting of patients with tubal infertility. After these two clinical presentations, there were two talks describing the regulation of ovarian PRAS in the cow ovary. The cow has proved to be an excellent experimental model for understanding the role of PRAS within the ovary. B. Brunswig-Spickenheier (Hamburg, Germany) showed that, in addition to LH, various growth factors and cytokines present within the ovary exert an autocrine and/or paracrine regulatory effect on the production of prorenin by bovine theta cells cultured in vitro. A.H. Nielson (Copenhagen, Denmark) described the interesting relationship between the follicular size on the one hand and the renin expression and AI1 receptor density in ovarian follicles on the other. A lengthy lunch-cum-poster session brought a number of interesting facets of ongoing research to light. They included recent development of new, specific,
10
01995,
reliable, and direct assays for prorenin. This is an important aspect for studies on tissue PRAS. Almost all extrarenal tissues expressing the renin gene secrete prorenin predominantly; hardly any active renin is released from such tissues. Therefore, availability of direct prorenin assays, bypassing the need for an activation of inactive prorenin and which is free from errors arising out of cryoactivation of prorenin in frozen samples, could increase the pace of research on tissue PRAS. Also, a number of posters discussed the role of tissue PRAS in the development of various endocrine malfunctions. The majority of posters presented pathophysiologic studies on PRAS in reproductive organs. Apart from the ovary, the uteroplacental unit is a major extrarenal source of prorenin. A. Poisner (Kansas City, KS, USA) lucidly explained the interactions among various second messenger systems, including, CAMP, protein kinase C, and Ca2+ (which he considers the most important second messenger) in the regulation of uteroplacental prorenin synthesis. K. Murakami (Tsukuba, Japan) described construction of chimeric mice containing both human renin &RN) and human angiotensinogen (hAG), in addition to their own endogenous PRAS and AI1 receptors. This was done by cross-mating individual lines of transgenes carrying either hRN or hAG. Female mice carrying hAG, when pregnant and carrying fetuses expressing hRN, died during the late pregnancy. This suggested that renin released in excess by the fetus could freely pass through the placenta and reach the maternal circulation. Apart from this important finding, Murakami also reported the construction of angiotensinogen gene knockout mice, which at the age of 3 weeks did not show any gross abnormality. The full significance of this observation in terms of the role of PRAS in cardiovascular function as well as in the local regulation of tissue function remains to be clarified. The possible role of prorenin in complications like retinopathy or nephropathy (microalbuminuria) associated with diabetes mellitus, was discussed by F.H.M. Derkx (Rotterdam, The Netherlands). From a lo-year-long prospective follow-up study, it appeared that an increased prorenin level in diabetic pa-
Elsevier
ScienceInc., 1043-2760/95/$9.50
SSDI
tients may be indicative of an imminent microvascular disease. Not all patients having these complications, however, had abnormally high levels of prorenin, thus limiting the predictive value of prorenin measurement in diabetes mellitus. An interesting inverse relationship between renin, angiotensinogen, AI and AI1 level in blood, and the severity of clinical symptoms in patients suffering from hymenoptera venom allergy was reported by K. Hermann (Chaska, MN, USA). Such patients had significantly lower renin, angiotensinogen, and AI and AI1 in the circulation than healthy volunteers, and following desensitization these values normalized. The final session was reserved for W. Ganong (San Francisco, CA, USA) to present concluding remarks and a general overview on the theme of the symposium, with many thought-provoking comments, in particular regarding the endocrine and neuroendocrine role of AII. Abstracts of the meeting have been published (Regulatory Peptides 53:133162, 1994). The complete proceedings of the symposium will be published by Plenum Press, New York, in early 1995. Apart from the symposium lectures and oral communications, which will be published as full and short papers, respectively, this volume will contain two additional sections. One section will contain comprehensive review articles on the molecular biology, physiology, and pharmacology of the renin-angiotensin system and the other will present chapters on various aspects of reproductive biology and endocrinology. This somewhat unusual but interesting combination of subjects was chosen for the book to serve as a reference for scientists and research students interested in either of the two areas. It should be of particular interest, however, to those researchers who work in areas where the two fields of interest merge. This meeting was generously supported by the Deutsche Forschungsgemeinschaft (DFG, Bonn, Germany), Gesellschaft zur Forderung Endokrinologischer Forschung (GFEF, Hamburg, Germany), the city of Hamburg, Institute for Hormone and Fertility Research at the University of Hamburg, and a number of commercial organizations. TEM
1043-2760(94)00091-H
TEM Vol. 6,No.I,1995
MEETING REPORT Hanseatic Endocrine Conference on Tissue Prorenin-Renin-Angiotensin Systems LocalRegulatory Actions in Reproductive and Endocrine Organs, Hamburg, July H-16,1994 Amal K. Mukhopadhyay
Over the past decade, substantial evidence has become available implicating tissue prorenin-renin-angiotensin systems (PRAS) in the local regulation of differentiated cell functions within the tissues where it is expressed. In contrast to a mixed enzyme-endocrine role classically ascribed to the renal renin-angiotensin system in the context of cardiovascular homeostasis, the tissue PRAS appears to play an autocrine, paracrine, or even an intracrine regulatory role. It has been well documented that PRAS is expressed in a wide variety of tissues, including reproductive and endocrine glands. We are now poised to take major strides toward understanding such fundamental issues as, for example, the tissue-specific regulation of the expression of PRAS, the nature of signal transduction pathways associated with various angiotensin II (AII) receptors, and the relevance of an aberrant expression of the tissue PRAS to pathophysiology encountered in a number of endocrine and reproductive organs. This symposium was organized as a satellite to the Third European Endocrinology Congress, Amsterdam, The Netherlands, to focus the discussion on current advances in these important aspects of tissue PRAS and to generate (Trends Endocrinol Metab 1995;6:8ideas on future research strategies. 10). The symposium started with a discussion on molecular aspects of the reninangiotensin system, including the regulation of tissue-specific expression of renin gene and the application of transgenic strategy. M. Paul (Berlin, Germany) briefly reviewed the available
data on gene expression of various components of prorenin-renin-angiotensin systems (PRAS) in several extrarenal organs. The link between the aberrant gene regulation of angiotensin-converting enzyme (ACE) and various cardiovascular abnormalities such as hypertension, myocardial infarction, and cardiac hypertrophy was particularly stressed by him. J. Mullins (Edinburgh, Amai K. Mukhopadhyayis at the Institutefor UK) discussed the benefits of transgenic Hormone and Fertility Research, University of Hamburg, Grandweg 64, D-22529 Ham- strategies to the understanding of the role of extrarenal PRAS. Transgenic rats burg, Germany. 8
01995,
EIsevIer Science Inc., 1043-2760/95/$9.50
SSDI
were produced by introducing a fusion transgene where a cDNA sequence encoding ren-2 was driven by a,-antitrypsin promoter. Such animals were characterized by extreme cardiac hypertrophy leading to an early death of the transgenic founders. Data from one that survived showed that the cardiac hypertrophy observed in these animals could be directly linked to the high level of extrarenal prorenin in their circulation, resulting from the expression of yen-2 gene solely in the liver. This once again underlined the importance of extrarenal prorenin with reference to the development of cardiovascular pathology. T. Voigtlander (Berlin, Germany) reported that intron I of the rat renin gene possibly contains a silencer involved in its tissue-specific expression. Such information on the transcriptional regulation of the renin gene are valuable for the understanding of both its normal and aberrant expression in extrarenal tissues. One of the complications of cardiovascular diseases associated with an increased circulatory renin level is the hypertrophy observed in cardiovascular and renal tissues, as just described. AI1 acts as a hypertrophic factor for smooth muscle cells in these tissues (W.A. Hsueh, Los Angeles, CA, USA; and G. Wolf, Hamburg, Germany). In cardiac tissues, according to W.A. Hsueh, AI1 acts on cardiac fibroblasts, which are richly endowed with AT, receptors to promote collagen and extracellular matrix production. Additionally, growth factors produced by such activated fibroblasts are responsible for the growth effects on cardiac myocytes in a paracrine manner. G. Wolf proposed that, in proximal tubular cells of the kidney, AII-induced cellular hypertrophy was mediated by transforming growth factor B (TGF@, since AI1 increased both the rate of transcription and the synthesis of TGFB in these cells and a neutralizing antiserum to TGFl3 could block the growth effects of AII. In cells like mesangial cells, however, where AI1 is mitogenic,
1043-2760(94)00091-H
TEM Vol. 6,No.1,1995
no induction of TGF@ was Thus, whether AI1 stimulation a cellular depends
hypertrophy on whether
observed. results in
or proliferation TGFfl is expressed
or not, which in turn is determined
by
thus appear to persist regarding the possible signal transduction cascades utilized by AT,. This remains, therefore, an
interesting
exploration.
area
requiring
Also, questions
further
such
as if
the ability of AI1 to induce certain trans-acting factors in a cell-specific manner. In the last talk of this session, using an interesting model of in-fieldstimulated guinea-pig atria, H. Brasch (Ltibeck, Germany) clearly showed that AI1 could stimulate the release of norepinephrine from sympathetic nerve endings by an activation of AT, receptors, and postulated that in this process AT, receptors somehow interact with c+ adrenoceptors. Knowledge of the molecular biology, physiology, and pharmacology of angiotensin receptors is fundamental to an understanding of how AI1 is able to bring about its diverse biologic effects in a variety of tissues. This was the theme of the third session of the first day, where interesting, but at times somewhat divergent, views were presented by S. Bottari (Grenoble, France) and T. Inagami (Nashville, TN, USA). According to the former, AI1 binds to two pharmacologically distinct receptor subtypes, AT, and AT,. Based on molecular cloning data, Inagami was able to subdivide the AT, type into subtypes AT,,, predominantly expressed in adrenal and pituitary, and which has 96% homology with AT,,, AT,, and is abundantly expressed in contractile and renal cells. Both subtypes belong to the seven transmembrane domain type receptor class acting via Gi or G, and being linked to diverse signaling pathways, including an inhibition of adenylate cyclase, activation of phospholipase A, C, or D, and the regulation of calcium channels. According to Bottari, AT, receptors, when activated in a ligand-specific manner, stimulate a phosphotyrosinephosphatase activity via a G-protein-independent mechanism. Such activation of phosphotyrosine phosphatase is, in an as yet unknown manner, linked to an inhibition of ANPreceptor-coupled guanylate cyclase activity. Expression cloning of the AT,
other classes or subclasses of AI1 receptors exist and if yes, where these are localized (at the cell surface or within the cell) will continue to engage scientific attention in the future. In this context, G. Vinson (London, UK), using a monoclonal antibody specifically directed against the N-terminal region of AT, receptors, showed that AT, receptors on adrenal
receptor yielded data which, according to Inagami, convincingly demonstrated that this receptor contains seven transmembrane domains and is linked to an activation of certain specific phosphotyrosine phosphatase, in a G-proteindependent manner. Some uncertainties
neuronal cell culture from the brain of SHR rats had not only more AT, receptor, but also a greater amount of the corresponding mRNA. Such high expression of AT, receptors in the brain of SHR rats may be responsible for a hyperactive renin-angiotensin system in the brain of
TEA4Vol. 6,No.1,1995
glomerulosa cells continuously cycle between the plasma membrane and intracellular locations. Stimulation of steroidogenesis by AII, however, does not require internalization of the receptors, as in cells where receptors were anchored to plasma membrane by the antibody, AI1 could still stimulate aldosterone production. J. Peters (Heidelberg, Germany), using the immunogold labeling technique, showed ultrastructural data that renin and ACE activities are localized within the rat adrenal mitochondria. Also, AII-binding sites were demonstrated on membrane preparations from purified mitochondria. Thus, a local role of AI1 at the mitochondrial level in regulating adrenal steroidogenesis was postulated. Such intracrine regulatory pathways are increasingly discussed with respect to tissue PRAS. Obviously, further studies are necessary to explore this interesting possibility. The next session was devoted to a discussion of the role of angiotensin as a neuroendocrine regulator, especially in the central nervous system. Tissue PRAS in the brain has been investigated by many laboratories to elucidate its role in the development of hypertension. Compared with normotensive WKY rats, SHR rats are known to possess a hyperactive renin-angiotensin system. Working on this model, M.K. Raizada (Gainesville, FL, USA) reported that the numbers of AT, receptors in neuronal cell cultures from the brain of SHR rats were vastly increased. Compared with WKY rats, the
01995, Elsevier Science Inc., 1043-2760/95/$9.50
SSDI
these animals. On the other hand, D.J. Campbell (Heidelberg, Australia) raised questions about the relative importance of brain AI1 in developing high blood pressure in SHR rats, since he could measure only extremely low levels of AI1 in the brain. The importance of brain angiotensin peptides in regulating the secretion of pituitary trophic hormones has been known for a long time. M.I. Phillips (Gainesville, FL, USA) described experiments designed to test the hypothesis that AI1 in the hypothalamus may be involved in the preovulatory luteinizing hormone (LH) surge. Using a model of ovariectomized estrogen-progesteroneprimed rats, it was shown that the LH surge in these animals was indeed preceded by a peak of hypothalamic AI1 level. This led Phillips to conclude that AI1 neurons in the hypothalamus may be implicated in the regulation of the LH surge. AI1 may not only perform in the regulation of trophic hormone release from the pituitary but may also influence the growth of the pituitary, since AI1 is known to be able to induce hyperplasia or cell proliferation in a number of tissues (see earlier discussion here). This question was addressed by M. Pawlikowski (Lodz, Poland). AI1 at subnanomolar concentrations stimulated radiolabeled thymidine incorporation in rat pituitary tumor cells. The cell proliferation indices were diminished by ACE inhibitors and the AI1 receptor antagonists losartan and PD123177. Three sessions on the second day were earmarked for discussions on the role of the renin-angiotensin system within reproductive tissues, such as ovary, testis, and the uteroplacental unit. F. Naftolin (New Haven, CT, USA) opened the second day with an in-depth review of the available evidence on the intraovarian regulatory role of PRAS. He underscored the role of AI1 produced within the ovary as an important regulator of ovulation. In addition, he highlighted the role of other bioactive angiotensin peptides like angiotensin III, angiotensin 1-7, and angiotensin 3-8. Clearly, more studies are necessary to define the intraovarian role of these peptides. Association of ovarian PRAS with follicular atresia in rat was convincingly demonstrated by A. Husain (Cleveland, OH, USA). Along with the expression of AT, receptors, the atretic granulosa cells start expressing
1043-2760(94)00091-H
9
ACE activity and the level of follicular fluid renin rises. He noted that ACE, but not AT, receptors, could be found also on nonatretic granulosa cells. A possible relevance of PRAS to male reproduction was discussed by A. Mukhopadhyay (Hamburg, Germany), who demonstrated the presence of prorenin in sperm-free human seminal fluid samples by a direct immunoradiometric assay, which did not require any prior enzymatic activation of prorenin. Western blotting procedure identified a 48-kD protein as prorenin in the seminal fluid, and there was a direct correlation between sperm density and the level of prorenin in the seminal fluid. The origin of prorenin in semen remains obscure, and major research efforts will be needed to discern if seminal PRAS is relevant for sperm function. Possible involvement of ovarian PRAS in the etiology of the ovarian hyperstimulation syndrome was discussed by R.S. Morris (Los Angeles, CA, USA), who proposed that the use of ACE inhibitors may ameliorate the severity of the disease by reducing vascular permeability. That the PRAS could be relevant to polycystic ovarian disease (PCOD) was suggested by I. Matinlauri (Turku, Finland), who observed a significantly greater elevation of total renin in circulation after gonadotropin stimulation in PCOD patients compared with a control group consisting of patients with tubal infertility. After these two clinical presentations, there were two talks describing the regulation of ovarian PRAS in the cow ovary. The cow has proved to be an excellent experimental model for understanding the role of PRAS within the ovary. B. Brunswig-Spickenheier (Hamburg, Germany) showed that, in addition to LH, various growth factors and cytokines present within the ovary exert an autocrine and/or paracrine regulatory effect on the production of prorenin by bovine theta cells cultured in vitro. A.H. Nielson (Copenhagen, Denmark) described the interesting relationship between the follicular size on the one hand and the renin expression and AI1 receptor density in ovarian follicles on the other. A lengthy lunch-cum-poster session brought a number of interesting facets of ongoing research to light. They included recent development of new, specific,
10
01995,
reliable, and direct assays for prorenin. This is an important aspect for studies on tissue PRAS. Almost all extrarenal tissues expressing the renin gene secrete prorenin predominantly; hardly any active renin is released from such tissues. Therefore, availability of direct prorenin assays, bypassing the need for an activation of inactive prorenin and which is free from errors arising out of cryoactivation of prorenin in frozen samples, could increase the pace of research on tissue PRAS. Also, a number of posters discussed the role of tissue PRAS in the development of various endocrine malfunctions. The majority of posters presented pathophysiologic studies on PRAS in reproductive organs. Apart from the ovary, the uteroplacental unit is a major extrarenal source of prorenin. A. Poisner (Kansas City, KS, USA) lucidly explained the interactions among various second messenger systems, including, CAMP, protein kinase C, and Ca2+ (which he considers the most important second messenger) in the regulation of uteroplacental prorenin synthesis. K. Murakami (Tsukuba, Japan) described construction of chimeric mice containing both human renin &RN) and human angiotensinogen (hAG), in addition to their own endogenous PRAS and AI1 receptors. This was done by cross-mating individual lines of transgenes carrying either hRN or hAG. Female mice carrying hAG, when pregnant and carrying fetuses expressing hRN, died during the late pregnancy. This suggested that renin released in excess by the fetus could freely pass through the placenta and reach the maternal circulation. Apart from this important finding, Murakami also reported the construction of angiotensinogen gene knockout mice, which at the age of 3 weeks did not show any gross abnormality. The full significance of this observation in terms of the role of PRAS in cardiovascular function as well as in the local regulation of tissue function remains to be clarified. The possible role of prorenin in complications like retinopathy or nephropathy (microalbuminuria) associated with diabetes mellitus, was discussed by F.H.M. Derkx (Rotterdam, The Netherlands). From a lo-year-long prospective follow-up study, it appeared that an increased prorenin level in diabetic pa-
Elsevier
ScienceInc., 1043-2760/95/$9.50
SSDI
tients may be indicative of an imminent microvascular disease. Not all patients having these complications, however, had abnormally high levels of prorenin, thus limiting the predictive value of prorenin measurement in diabetes mellitus. An interesting inverse relationship between renin, angiotensinogen, AI and AI1 level in blood, and the severity of clinical symptoms in patients suffering from hymenoptera venom allergy was reported by K. Hermann (Chaska, MN, USA). Such patients had significantly lower renin, angiotensinogen, and AI and AI1 in the circulation than healthy volunteers, and following desensitization these values normalized. The final session was reserved for W. Ganong (San Francisco, CA, USA) to present concluding remarks and a general overview on the theme of the symposium, with many thought-provoking comments, in particular regarding the endocrine and neuroendocrine role of AII. Abstracts of the meeting have been published (Regulatory Peptides 53:133162, 1994). The complete proceedings of the symposium will be published by Plenum Press, New York, in early 1995. Apart from the symposium lectures and oral communications, which will be published as full and short papers, respectively, this volume will contain two additional sections. One section will contain comprehensive review articles on the molecular biology, physiology, and pharmacology of the renin-angiotensin system and the other will present chapters on various aspects of reproductive biology and endocrinology. This somewhat unusual but interesting combination of subjects was chosen for the book to serve as a reference for scientists and research students interested in either of the two areas. It should be of particular interest, however, to those researchers who work in areas where the two fields of interest merge. This meeting was generously supported by the Deutsche Forschungsgemeinschaft (DFG, Bonn, Germany), Gesellschaft zur Forderung Endokrinologischer Forschung (GFEF, Hamburg, Germany), the city of Hamburg, Institute for Hormone and Fertility Research at the University of Hamburg, and a number of commercial organizations. TEM
1043-2760(94)00091-H
TEM Vol. 6,No.I,1995