Adrenal medulla of AS/AGU rats: a histological and immunohistochemical study.

BACKGROUND: The outcome of the autograft therapy for Parkinson's disease including autologous cells from adrenal medulla was disappointing. This could be attributed to the pathological process in Parkinson's disease affecting cells of the adrenal medulla. This study was performed to investigate the histopathological changes in the adrenal medulla of AS/AGU rat, a model of Parkinson's disease, in comparison with Albino Swiss (AS) rats. MATERIALS AND METHODS: A total of 24 male AS rats were divided into four groups, each of 6 animals: AS W1 - AS rats aged 1 week; AS adult - AS adult rats; AS/ /AGU W1 - AS/AGU rats aged 1 week; and AS/AGU adult - AS/AGU adult rats. The rats were sacrificed and the adrenal glands were dissected and processed for histological staining with haematoxylin and eosin and periodic acid Schiff and for immunohistochemical staining for S100 protein, ubiquitin and tyrosine hydroxylase. RESULTS: The histological investigation of the adrenal medulla of AS/AGU rats showed vascular congestion, inflammatory cellular infiltration, pyknotic nuclei, necrotic chromaffin cells and medullary inclusion bodies. The immunohistochemical investigation of AS/AGU rats showed a statistically significant decrease in the expression of S100 protein, ubiquitin and tyrosine hydroxylase compared to AS rats. CONCLUSIONS: The histological and immunohistological changes in the adrenal medulla could explain the failure of outcome of adrenal autograft therapy in Parkinson's disease.

Role of VEGF-A and its receptors in sporadic and MEN2-associated pheochromocytoma.

Pheochromocytoma (PHEO), a rare catecholamine producing tumor arising from the chromaffin cells, may occurs sporadically (76%-80%) or as part of inherited syndromes (20%-24%). Angiogenesis is a fundamental step in tumor proliferation and vascular endothelial growth factor (VEGF-A) is the most well-characterized angiogenic factor. The role of angiogenic markers in PHEO is not fully understood; investigations were therefore made to evaluate the expression of VEGF-A and its receptors in PHEO and correlate to clinical parameters. Twenty-nine samples of PHEO were evaluated for VEGF-A, VEGF receptor-1 (VEGFR-1) VEGFR-2 expression and microvessel density (MVD) by immunohistochemistry. Clinical data were reviewed in medical records. The mean age of patients was 38±14 years, and 69% were woman. VEGF-A, VEGFR-1 and VEGFR-2 staining were detected in nearly all PHEO samples. No significant correlation was observed between VEGF-A, VEGFR-1, VEGFR-2 expression or MVD and age at diagnosis, tumor size or sporadic and hereditary PHEO. However, the levels of expression of these molecules were significantly higher in malignant PHEO samples (p=0.027, p=0.003 and p=0.026, respectively).VEGF-A and its receptors were shown to be up-regulated in malignant PHEO, suggesting that these molecules might be considered as therapeutic targets for unresectable or metastatic tumors.

Adrenal glands of slaughtered bulls, heifers and cows: a histological study.

The study involved histological and immunohistochemical examinations of the adrenal glands of healthy slaughtered cattle. Glands of 13 bulls, 10 heifers and 10 cows were examined. The following histological findings were observed: Unequal thickness of connective capsule and nodular formations of the zona glomerulosa (ZG), eosinophilic granules in cells of the ZG, globoid arrangement of the zona fasciculata, nodules or pegs of cortical tissue in the medulla, mutual interlacing of superficial and deep zones of the medulla, proliferation of cortical or medullary cells into the blood vessels wall situated in the medulla and focal inflammatory infiltrates. Cortical cells and noradrenalin-secreting (N) cells in the medulla expressed cytoplasmic positivity of S100 protein. Both adrenalin (A) cells and N cells were positive in synaptophysin. The majority of the cells in the cortex and in the medulla displayed were positive for chromogranin A. Electron microscopy showed structureless, electrondense particles of varying size and shape, mostly displaying the having mostly character of secretory granules.

Nicotine-evoked cytosolic Ca(2+) increase and cell depolarization in capillary endothelial cells of the bovine adrenal medulla.

Endothelial cells are directly involved in many functions of the cardiovascular system by regulating blood flow and blood pressure through Ca(2+) dependent exocitosis of vasoactive compounds. Using the Ca(2+) indicator Fluo-3 and the patch-clamp technique, we show that bovine adrenal medulla capillary endothelial cells (B AMCECs) respond to acetylcholine (ACh) with a cytosolic Ca(2+) increase and depolarization of the membrane potential (20.3+/-0.9 mV; n=23). The increase in cytosolic Ca(2+) induced by 10microM ACh was mimicked by the same concentration of nicotine but not by muscarine and was blocked by 100 microM of hexamethonium. On the other hand, the increase in cytosolic Ca(2+) could be depressed by nifedipine (0.01 -100 microM) or withdrawal of extracellular Ca(2+). Taken together, these results give evidence for functional nicotinic receptors (nAChRs) in capillary endothelial cells of the adrenal medulla. It suggests that nAChRs in B AMCECs may be involved in the regulation of the adrenal gland's microcirculation by depolarizing the membrane potential, leading to the opening of voltage-activated Ca(2+) channels, influx of external Ca(2+) and liberation of vasoactive compounds.

Nicotine-evoked cytosolic Ca2+ increase and cell depolarization in capillary endothelial cells of the bovine adrenal medulla

Endothelial cells are directly involved in many functions of the cardiovascular system by regulating blood flow and blood pressure through Ca2+ dependent exocitosis of vasoactive compounds. Using the Ca2+ indicator Fluo-3 and the patch-clamp technique, we show that bovine adrenal medulla capillary endothelial cells (B AMCECs) respond to acetylcholine (ACh) with a cytosolic Ca2+ increase and depolarization of the membrane potential (20.3±0.9 mV; n=23). The increase in cytosolic Ca2+ induced by 10µM ACh was mimicked by the same concentration of nicotine but not by muscarine and was blocked by 100 µM of hexamethonium. On the other hand, the increase in cytosolic Ca2+ could be depressed by nifedipine (0.01 -100 µM) or withdrawal of extracellular Ca2+. Taken together, these results give evidence for functional nicotinic receptors (nAChRs) in capillary endothelial cells of the adrenal medulla. It suggests that nAChRs in B AMCECs may be involved in the regulation of the adrenal gland's microcirculation by depolarizing the membrane potential, leading to the opening of voltage-activated Ca2+ channels, influx of external Ca2+ and liberation of vasoactive compounds.

[Morphometric parameters of rat adrenals in the dynamics of general hypothermia].

In this study, some morphometric parameters of adrenals in outbred albino male rats were compared in the dynamics of general hypothermia at the ambient temperature of -18 degrees C. It was shown that in the course of general hypothermia, the increase of blood vessel relative volume in zona reticularis and in adrenal medulla was accompanied by the augmentation of nucleus size of adrenocorticocytes (mainly in zona fasciculata). Zona fasciculata reacted with a significant increase of blood vessel relative volume during the 1st hour of cold exposure, henceforth the parameters remained unchanged. Blood vessel relative volume in the left adrenal was found to significantly exceed that in the right adrenal. In zona glomerulosa of the left adrenal, blood vessel relative volume was reduced, while that one in zona glomerulosa of the right adrenal remained unchanged during the whole experiment. Volume density of mitochondria in the endocrine cells of zona fasciculata was found to increase, this effect being more pronounced in the right adrenal as compared to the left one. In the cells of both glands, the volume density of lipid inclusions was gradually reduced, while the relative volume of nucleoli was variable and there were no statistically significant changes detected during the course of hypothermia.

Active stress kinases in proliferating endothelial cells associated with cytoskeletal structures.

It has become increasingly clear that stress-activated protein kinases have cytoplasmic substrates in addition to well-established transcription factor substrates in cell nuclei. The present study documented specific cytoplasmic locations of these enzymes in proliferating vascular cells. Immunofluorescent staining for active c-jun NH2-terminal kinase (JNK), the precipitation of JNK with microfilaments, and the loss of fiber-associated active JNK after cytochalasin treatment, but not nocodazole treatment, together indicate that active JNK is associated with stress fibers. The lack of complete scaffold colocalization and the total lack of immediate upsteam kinase colocalization along with the inability of JNK inhibitors to alter JNK-microfilament associations suggest that the microfilament association is not simply involved in enzyme activation. In addition, active p38 was found along with vinculin in focal adhesions. Although the p38 in focal adhesions could also be disrupted by cytochalasin treatment, it remained stable after nocodazole treatment. These results support the hypothesis that vascular cell stress kinase enzymes are important for signal transduction in the cytoplasm. The localization of active stress-activated protein kinases to specific cytoskeletal structures in proliferating cells suggests that subsets of these enzymes are involved in signal transduction to and/or from the cytoskeleton under conditions that include vascular cell proliferation.

Nitric oxide inhibits the bradykinin B2 receptor-mediated adrenomedullary catecholamine release but has no effect on adrenal blood flow response in vivo.

The role of nitric oxide (NO) in bradykinin (BK)-induced adrenal catecholamine secretion still remains obscure. The present study was to investigate whether an inhibition of NO synthase with N(omega)-nitro-L-arginine methyl ester (L-NAME) would modulate BK-induced adrenal catecholamine secretion (ACS) and adrenal vasodilating response (AVR) in anesthetized dogs. Plasma catecholamine concentrations were determined with an HPLC coupled with an electrochemical detector. All drugs were locally administered to the left adrenal gland via intra-arterial infusion. BK dose-dependently increased both ACS and AVR. Hoe-140, a selective B(2) antagonist, significantly blocked the BK-induced increases in both ACS and AVR. In the presence of L-NAME, the BK-induced ACS was significantly enhanced, while the simultaneous AVR remained unaffected. These results suggest that the both BK-induced ACS and AVR are primarily mediated by B(2) receptors in the canine adrenal gland. Our results also suggest that the enhanced ACS in response to BK in the presence of L-NAME may have resulted from a specific inhibition of NO formation in the adrenal gland. It is concluded that the BK-induced NO may play an inhibitory role in the B(2)-receptor-mediated mechanisms regulating ACS, while it may not be implicated in the B(2)-receptor-mediated AVR under in vivo conditions.

[Effect of hypoxic stress on the medullary arteries of the adrenal glands]. / Uticaj hipoksicnog stresa na medularne arterije nadbubrezne zlezde.

INTRODUCTION: It is known that suprarenal glands consist of two parts: cortex and medulla. Functional properties of circulation in these two parts are still unknown. This is a very important problem, but only a few studies have dealt with it, whereas full attention should be paid to the problem in future researches. MATERIAL AND METHODS: The experiment included 100 sexually experienced male rats which were exposed to hypoxia in testing chambers imitating conditions at 7000 m above sea level. In order to study blood vessels, we used a mixture of indian ink and 10% solution of gelatin and injected it into the left heart ventricle. RESULTS AND DISCUSSION: The most outstanding finding pointed to existence of wide ischemic areas in the outer portion of zona fasciculata, along with simultaneous strong dilatation and increased number of medullary arteries. Therefore, a conclusion can be made that the number of opened medullary arteries in the suprarenal gland depends on functional conditions and needs of the organism. The method used to present our results showed that vascular network of the suprarenal glands changed dynamically under experimental conditions. CONCLUSION: The obtained results point to functional accommodation of vascular network of suprarenal glands affected by stress, whereas medullary arteries are of predominantly functional character.

Cotinine inhibits catecholamine release evoked by cholinergic stimulation from the rat adrenal medulla.

The aim of the present study was to clarify whether cotinine affects the release of catecholamines (CA) from the isolated perfused rat adrenal gland, and to establish the mechanism of its action, in comparison with the response of nicotine. Cotinine (0.3-3 mM), when perfused into an adrenal vein for 60 min, inhibited CA secretory responses evoked by ACh (5.32 mM), DMPP (a selective neuronal nicotinic agonist, 100 microM for 2 min) and McN-A-343 (a selective muscarinic M1-agonist, 100 microM for 2 min) in dose- and time-dependent manners. However, cotinine did not affect CA secretion by high K+ (56 mM). Cotinine itself also failed to affect basal CA output. Furthermore, in the presence of cotinine (1 mM), CA secretory responses evoked by Bay-K-8644 (an activator of L-type Ca2+ channels, 10 microM) and cyclopiazonic acid (an inhibitor of cytoplasmic Ca2+-ATPase, 10 microM) were relative time-dependently attenuated. However, nicotine (30 microM), given into the adrenal gland for 60 min, initially rather enhanced CA secretory responses evoked by ACh and high K+, followed by the inhibition later, while it time-dependently depressed the CA release evoked by McN-A-343 and DMPP. Taken together, these results suggest that cotinine inhibits greatly CA secretion evoked by stimulation of cholinergic (both nicotinic and muscarinic) receptors, but does fail to affect that by the direct membrane-depolarization. It seems that this inhibitory effect of cotinine may be exerted by the cholinergic blockade, which is associated with blocking both the calcium influx into the rat adrenal medullary chromaffin cells and Ca2+ release from the cytoplasmic calcium store. It also seems that there is a big difference in the mode of action between cotinine and nicotine in the rat adrenomedullary CA secretion.

Effects of acute administration of ethanol on the rat adrenal cortex.

OBJECTIVE: The purpose of this study was to investigate the effect of a single dose of ethanol on rat adrenal cortex and to determine whether the estrous cycle can influence this effect of ethanol. METHOD: Adult female Wistar rats showing proestrus or diestrus Day 1 (n = 12) were treated intraperitoneally with ethanol (4 g/kg body weight). Untreated (n = 15) and saline-injected (n = 14) rats were used as controls. The animals were sacrificed by decapitation 0.5 hour after ethanol administration. Stereological analysis was performed on paraffin sections of adrenal glands stained with AZAN, and the following parameters were determined: absolute volume of the zona glomerulosa, the zona fasciculata and the zona reticularis, numerical density, volume and the mean diameter of adrenocortical cells and of their nuclei, and diameter and length of capillaries. RESULTS: The diameter and volume of adrenocortical cells in the zona fasciculata and the zona reticularis were significantly increased by acute ethanol treatment at proestrus. In the same group of animals, a single dose of ethanol induced significant decrease in numerical density of adrenocortical cells and of their nuclei in all three zones. Increased length of capillaries of the zona fasciculata as well as enhanced level of serum corticosterone was found in ethanol-treated rats at both phases of the estrous cycle, proestrus and diestrus Day 1. CONCLUSIONS: The obtained results indicate that a single dose of ethanol activates adrenal cortex in female rats and that the effect is more pronounced on morphometric parameters at proestrus.

Role of PAC(1) receptor in adrenal catecholamine secretion induced by PACAP and VIP in vivo.

The present study was conducted to investigate the functional implication of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I (PAC(1)) receptor in the adrenal catecholamine (CA) secretion induced by either PACAP-27 or vasoactive intestinal polypeptide (VIP) in anesthetized dogs. PACAP-27, VIP, and their respective antagonists were locally infused to the left adrenal gland via the left adrenolumbar artery. Plasma CA concentrations in adrenal venous and aortic blood were determined by means of a high-performance liquid chromatograph coupled with an electrochemical detector. Adrenal venous blood flow was measured by gravimetry. The administration of PACAP-27 (50 ng) resulted in a significant increase in adrenal CA output. VIP (5 microg) also increased the basal CA secretion to an extent comparable to that observed with PACAP-27. In the presence of PACAP partial sequence 6--27 [PACAP-(6--27); a PAC(1) receptor antagonist] at the doses of 7.5 and 15 microg, the CA response to PACAP-27 was attenuated by approximately 50 and approximately 95%, respectively. Although the CA secretagogue effect of VIP was blocked by approximately 85% in the presence of PACAP-(6--27) (15 microg), it remained unaffected by VIP partial sequence 10--28 [VIP-(10--28); a VIP receptor antagonist] at the dose of 15 microg. Furthermore, the CA response to PACAP-27 did not change in the presence of the same dose of VIP--(10--28). The results indicate that PACAP-(6--27) diminished, in a dose-dependent manner, the increase in adrenal CA secretion induced by PACAP-27. The results also indicate that the CA response to either PACAP-27 or VIP was selectively inhibited by PACAP-(6--27) but not by VIP-(10--28). It is concluded that PAC(1) receptor is primarily involved in the CA secretion induced by both PACAP-27 and VIP in the canine adrenal medulla in vivo.

Activation of sympathoadrenomedullary system increases pulmonary nitric oxide production in the rabbit.

Nitric oxide (NO) is continuously produced in the lung and can be measured in exhaled gas of different species. To investigate a possible neuro-humoral regulation of pulmonary NO production in vivo we injected veratrine, an activator of Na(+) channels known to activate the sympathoadrenal system, in anaesthetized, mechanically ventilated and laparotomized rabbits. Exhaled NO concentration increased by 38+/-3% when plasma adrenaline rose from 12.3+/-3.1 to 49.5+/-10.7 pmol ml(-1) in response to veratrine (500 microg kg(-1), i.v.). Pretreatment with atenolol, a beta(1)-adrenoceptor antagonist (1 mg kg(-1)), or bilateral ligation of adrenal blood vessels inhibited the increase in exhaled NO in response to veratrine. Atenolol also decreased basal NO, suggesting an endogenous regulation of pulmonary NO by adrenaline. Neither phentolamine (1 mg kg(-1)), atropine (1 mg kg(-1)) nor vagotomy inhibited the veratrine-induced pulmonary NO production. These results suggest a role of the sympathoadrenal system in the regulation of pulmonary NO production.

[Determination of catecholamine secreted from rat adrenal medulla by in vivo voltammetry].

In vivo voltammetry using carbon fiber electrodes was employed to determine the catecholamine secreted from the adrenal medulla. The basal level of catecholamine in the medulla was between 0.1 and 0.5 micromol/L, but it increased markedly up to 5 30 micromol/L when the gland was made ischemic. The spontaneous release of catecholamine from the medulla was observed in both normal and ischemia conditions, but the frequency and amplitude of spontaneous release were increased significantly by ischemia. This implied that the animal was under serious stress condition. The above results also show that acetylcholine can induce the secretion of catecholamine in a dose-dependent manner.

Adrenal microvascularization in the common tree shrew (Tupaia glis) as revealed by scanning electron microscopy of vascular corrosion casts.

The blood supply of the adrenal gland in the common tree shrew (Tupaia glis) was studied by use of transmission electron microscopy and vascular corrosion cast/scanning electron microscopy techniques. It was found that the gland receives its blood supply from branches of the inferior phrenic, aorta and renal arteries. Upon reaching the gland, these arteries divide into cortical and medullary arteries. The cortical arteries give rise to the subcapsular capillary plexuses which partially enclose the clusters of cells in the zona glomerulosa (ZG) and appear as lobular-like microvascular networks before running among the cellular cords in the zona fasciculata (ZF) and zona reticularis (ZR). It was noted that the capillaries in ZG and ZR are with more anastomoses than those in the ZF. Capillaries from the ZR become the sinusoidal capillaries in the adrenal medulla before proceeding to the peripheral radicles of the central vein. The medullary arteries penetrate the adrenal cortex and occasionally give off small branches to supply the inner cortex, especially the ZR. Their main branches break up into small or conventional capillaries in the adrenal medulla. These capillaries drain the blood into the peripheral radicles of the central vein and medullary collecting veins which proceed further into a very large central vein. The present findings illustrate that the adrenal medulla receives two blood supplies that yield somewhat different influences upon the adrenal medulla. The portal blood vessel could not be illustrated in the tree shrew adrenal gland.

The adrenomedullary venous vasculature as a target for endothelins: comparison of the porcine and bovine central adrenomedullary veins.

The functional properties of the isolated porcine and bovine central adrenomedullary veins were compared, with emphasis on the active tension responses to high K+, endothelin-1 (ET-1) and neuropeptide Y (NPY). In the porcine vein, the contraction evoked by ET-1 was 4--5-fold higher than with high K+, as in the bovine vein. The potencies for ET-1 were similar in ring and strip preparations of the porcine vein, with EC50 values 5--7-fold higher than in the bovine vein. In preparations previously exposed to ET-1 the contractions evoked by high K+ and NPY were potentiated and facilitated, respectively,. However, only in the porcine vein was the ET-1 contraction sustained. This contraction was effectively relaxed by milrinone, indicating a role for cGMP inhibited cyclic nucleotide phosphodiesterase in the sustained contraction. Caffeine and forskolin were also effective relaxants of contractions evoked by ET-1 in both veins, suggesting relaxation by elevated levels of cAMP. The K(+)-contracted porcine, but not bovine, vein was relaxed by acetylcholine (ACh) and vasointestinal polypeptide in a concentration-dependent manner, indicating species differences with respect to signal transduction leading to increases in cyclic nucleotides. In conclusion, these results demonstrate that ET-1 is the main constrictor of the porcine central adrenomedullary vein, with significant species differences in mode of contraction and relaxation. These findings suggest roles for the endogeneously released ET-1 and NPY in regulation of venous contractility within the adrenal gland of mammals.

The adrenal medulla as a wet sponge: a role for the intramedullary venous vasculature?

Synchronous contractions in the intramedullary venous vasculature have been postulated to assist in the discharge of hormones from the stimulated adrenal medulla in a manner analogous to the squeezing of a wet sponge. This study reports on two experimental approaches to support the hypothesis that contractions in the venous vasculature may contribute to the hormonal efflux. Firstly, the bovine adrenal medulla was perfused retrogradely via the bovine central adrenomedullary vein end changes in the vascular volume were assessed as changes in wet weight of the perfused tissue. Stimulation with acetylcholine and carbachol resulted in repetitive, transient weight losses, suggesting cholinergically mediated reductions in the vascular volume. Secondly, the contractile properties of the longitudinal layers of smooth muscle cells in the intramedullary venous system were characterized, using the bovine central adrenomedullary vein as a model. The results showed that the longitudinal layers of this vein were, similarly to the circular layers, selectively contracted by endothelin-1 via an ETA-like receptor, by neuropeptide Y and by membrane depolarization (high K+). However, the vein was insensitive to electrical stimulation acetylcholine and carbacho, as well as to catecholamines. These results suggest neuropeptide Y, released from the cholinergically stimulated chromaffin cells, as the most likely mediator of stimulus-evoked synchronous contractions of the venous vasculature in the bovine adrenal medulla. Together, these experiments provide support for the 'wet sponge' hypothesis for hormonal discharge from the adrenal gland.

Ecto-enzymatic hydrolysis of diadenosine polyphosphates by cultured adrenomedullary vascular endothelial cells.

We investigated the extracellular degradation of diadenosine polyphosphates (ApnA) by cultured adrenomedullary endothelial cells using fluorogenic analogs of ApnA, the di(1,N6-ethenoadenosine) 5',5"'-P1,Pn-polyphosphates [epsilon-(ApnA)]. Kinetic parameters of epsilon-(ApnA) cleavage and effects of pH, ions, and inhibitors were determined by continuous fluorometric assays, using suspensions of endothelial cells grown on Cytodex-1 microspheres. Ecto-enzyme kinetic parameters for epsilon-(Ap3A), epsilon-(Ap4A), and epsilon-(Ap5A) hydrolysis are as follows: Michaelis-Menten constants of 0.39 +/- 0.07, 0.42 +/- 0.09, and 0.37 +/- 0.05 microM respectively, and maximal velocities of 26.1 +/- 6.8, 74.2 +/- 16.4, and 24.4 +/- 3.4 pmol.min-1.10(6) cells-1, respectively. ApnA and guanosine 5',5"'-P1,P4-tetraphosphate behave as competitor substrates of epsilon-(Ap4A) hydrolysis. The ectoenzyme is activated by Mg2+ and Mn2+ and inhibited by Ca2+, F-, adenosine 5'-tetraphosphate, adenosine 5'-O-(3-thiotriphosphate), and suramin. Optimum pH is around 9.0. High-performance liquid chromatography analysis reveals that the ecto-enzyme hydrolyzes epsilon-(ApnA) to give epsilon-adenosine-5'(n-1)-phosphate and epsilon-AMP, which are then further catabolized up to epsilon-adenosine via the membrane-bound nucleotidase system ecto-ATPase, ecto-ADPase (or apyrase), and ecto-5'-nucleotidase. The endothelial ecto-diadenosine polyphosphate hydrolase studied here exhibits different kinetic parameters and sensitivity to ions with respect to the enzyme from the tissue-related neurochromaffin cells. These different properties may be important in the extracellular signaling by ApnA.

The thrombin receptor in adrenal medullary microvascular endothelial cells is negatively coupled to adenylyl cyclase through a Gi protein.

The effects of thrombin on adenylyl cyclase activity were examined in rat adrenal medullary microvascular endothelial cells (RAMEC). Confluent RAMEC monolayers were stimulated for 5 min with cAMP-generating agents in the absence and presence of thrombin, and intracellular cAMP was measured with a radioligand binding assay. Thrombin (0.001-0.25 U/ml) dose-dependently inhibited IBMX-, isoproterenol- and forskolin-stimulated cAMP accumulation. A peptide agonist of the thrombin receptor, gamma-thrombin, and the serine proteases trypsin and plasmin, also inhibited agonist-stimulated cAMP levels, while proteolytically inactive PPACK- or DIP-alpha-thrombins were without effect. Moreover, the thrombin inhibitor hirudin abolished the inhibitory effect of thrombin but not of the peptide agonist. These results suggest that the inhibitory action of thrombin on cAMP accumulation is mediated by a proteolytically-activated thrombin receptor. The inhibitor of G(i)-proteins pertussis toxin abolished the inhibitory effect of thrombin on isoproterenol- or IBMX-stimulated cAMP production, while the phorbol ester PMA partly impaired it. The protein kinase C inhibitors staurosporine or H7 and the intracellular Ca2+ chelator BAPTA-AM were without effect. Collectively, our data suggest that the thrombin receptor in RAMEC is negatively coupled to adenylyl cyclase through a pertussis toxin-sensitive G(i)-protein.

Biphasic estrogen response on bovine adrenal medulla capillary endothelial cell adhesion, proliferation and tube formation.

Abnormal angiogenesis underlies many pathological conditions and is critical for the growth and maintenance of various types of tumors, including hormone-dependent cancers. Since estrogens are potent carcinogens in humans and rodents, and are involved in regulating angiogenesis, this study was designed to examine the effect of estrogen on the behavior of an established bovine capillary endothelial cell line, a simple and physiologically relevant model of the capillary wall. The results demonstrate that 17beta-estradiol (E2), at different conditions, exerts both stimulatory and inhibitory effects on endothelial cell adhesion, proliferation and tube formation in vitro. Utilizing a cellular attachment assay, chronic exposure to nanomolar concentrations of E2 (i.e. 1 and 10 nM) increased endothelial cell adhesion significantly compared to vehicle treated controls. Cellular adhesion was inhibited by micromolar concentrations of E2. Cell count, PCNA immunohistochemistry and Western blot analysis demonstrated enhanced cell proliferation at low E2 concentration in estrogen-deplete medium. Inhibition of cellular proliferation was observed in both estrogen-replete and deplete medium at higher E2 concentrations (i.e. 1 and 10 microM). Furthermore, in vitro tube formation increased up to 3.0 fold in the presence of 10 nM and higher E2 concentrations. The present observations indicate that in vitro regulation of capillary endothelial cell adhesion, proliferation and capillary tube formation by estrogen, are dose dependent.