Role of endogenous prostaglandin E2 in erythropoietin production and dome formation by human renal carcinoma cells in culture.

Studies were carried out on the role of endogenous prostaglandin E2 (PGE2) in erythropoietin (Ep) production and dome formation in primary monolayer cultures of a human renal carcinoma from a patient with erythrocytosis that has been serially transplanted into BALB/c athymic nude mice. The metabolism of [14C]arachidonic acid (14C-AA) by cultured renal carcinoma cells, which were plated in 25-cm2 flasks at a density of 2 X 10(4) cells/cm2 and grown for 6, 12 (confluence, 13 X 10(4) cells/cm2), 16, 24, and 30 d in Eagle's minimum essential medium (MEM) supplemented with 10% fetal bovine serum, was examined by using radiometric thin-layer chromatography (TLC). TLC revealed PGE2 to be the major metabolite of 14C-AA produced by the cultured cells throughout the 30 d of cultivation. In addition, the cultured cells at each time period were incubated for 24 h in 5 ml of serum-free Eagle's MEM and the levels of PGE2 and Ep in the incubated media were measured via radioimmunoassay. PGE2 levels in the serum-free media incubated with the cultured cells grown for 6 d were significantly (P less than 0.001) elevated (174 +/- 2.5 pg/ml, n = 5), compared with the unincubated control media (1.5 +/- 0.19 pg/ml, n = 5) and gradually decreased at each time period to 97.6 +/- 4.4 pg/ml (n = 5) at 30 d. On the other hand, the levels of Ep in the incubated media of the cells grown for 6 d were 11.5 +/- 0.52 mU/ml (n = 5) compared with 7.6 +/- 0.62 mU/ml (n = 5) in the control media. However, after the cultured cells became confluent, the levels of Ep in the incubated media showed a marked increase to 222.9 +/- 5.26 mU/ml (n = 5) at 30 d of cultivation. Multicellular hemicysts (domes) developed after the cultured cells reached confluence and their numbers increased with increasing time in confluence in parallel with the increase in Ep. Meclofenamate (MF) (3 X 10(-6)-3 X 10(-5) M), a prostaglandin synthesis inhibitor, produced a significant dose-related decrease in PGE2, Ep, and dome formation without producing a significant effect on cell viability in the 30-d cells. This inhibitory effect of MF on Ep production and dome formation was completely abolished by the addition of 10(-8) M PGE2 to the incubation medium. In conclusion, endogenous PGE2 plays an important role in supporting and/or stimulating Ep production and dome formation in cultured renal carcinoma cells.

Modulation of prostaglandin biosynthesis in hypoxic murine mammary adenocarcinoma cells by misonidazole.

Resistance of hypoxic cells to radiation and chemotherapy remains a major limitation to effective therapy of solid tumors. Misonidazole, a 2-nitroimidazole analogue, has been studied extensively as a radiosensitizer of hypoxic cells and has been shown to undergo bioreductive metabolism to exert preferential cytotoxicity against hypoxic cells. We have investigated the effects of misonidazole on the biosynthesis of prostaglandins (PGs) in a murine mammary adenocarcinoma cell line (No. 4526) under aerobic and hypoxic conditions in attempts to exploit modulation of PG levels under hypoxia as a means of improving therapeutic approaches for the treatment of solid tumors. We report a time-dependent inhibition of PG biosynthesis by the suspended cells under hypoxia induced by flushing sealed vials with N2 (1.5 liters/min). After 30 min of hypoxia, PG formation was inhibited by 50%. Indomethacin was able to further inhibit the PG formation in a concentration-dependent manner under hypoxia. Misonidazole, however, selectively increased the PGE2 biosynthesis under hypoxia by 49% at 100 microM. This increase was concentration dependent over the range of 25 to 100 microM and was blocked by indomethacin (0.1 microM). Imidazole, the heterocyclic moiety in misonidazole without the nitro function, had no effect on PG biosynthesis at these concentrations. These data suggest that arachidonic acid metabolism is sensitive to the differential oxygen levels which exist within solid tumors and that PG levels may be modulated by electron-affinic agents in hypoxic tumor cells.

Evidence for a bidirectional prostaglandin endoperoxide shunt between platelets and the bovine coronary artery.

While platelets have been shown to be capable of supplying prostaglandin (PG) H2 to endothelial cells in culture for PGI2 synthesis, endothelial cells have been shown unable to supply PGH2 to platelets for thromboxane (TX) A2 synthesis. We incubated rings of the bovine coronary artery (BCAR) with human platelets treated with aspirin (to inhibit cyclooxygenase) or CGS 13080 (to inhibit TXA2 synthase) in the presence of 20 microM arachidonic acid. BCAR, with damaged endothelium, produced significantly less PGI2 than that with intact endothelium. However, co-incubation with CGS 13080-treated platelets resulted in an increase in PGI2 independent of endothelium, demonstrating a shunt of PGH2 from platelets to BCAR. Co-incubation of BCAR with aspirin-treated platelets resulted in a net increase in TXA2 demonstrating a shunt of PGH2 from BCAR to platelets. Employing [14C]PGH2 as substrate, BCAR with and without intact endothelium produced similar amounts of 6-keto-[14C]PGF1 alpha. Likewise, homogenates (50 micrograms protein) of intimal and subintimal regions of BCAR and BCAR converted similar amounts of PGH2 to 6-keto-PGF1 alpha. These data suggest that vascular production of PGH2 is more dependent on an intact endothelium than is the conversion of PGH2 to PGI2. These data also suggest a potential for a bidirectional exchange of PGH2 between platelets and vascular wall during platelet-vascular wall interactions.

Concentration-activity profile of the modulation of cyclooxygenase product formation by reduced glutathione in microsomal fractions from the goat lung.

Age-related changes in pulmonary formation of arachidonic acid (AA) metabolites are thought to play an important role in regulating cardiopulmonary function. This study addresses the potential role of reduced glutathione (GSH) in modulating cyclooxygenase product formation in the developing lung. Prostaglandin H2 (PGH2) metabolism was studied in microsomal fractions isolated from the lungs of unventilated fetal, neonatal and adult goats. GSH-dependent PGH2 to PGE2 isomerase activity in microsomal fractions from the perinatal (fetal and neonatal) goat lung was not saturable with respect to GSH and can respond to changes in GSH concentration over the range of 0.01 to 30 mM, which encompasses the full range the intracellular GSH levels reported in the literature. However, in fractions from the adult, a lower rate of PGE2 formation is observed at higher GSH concentrations. In addition, the tissue levels of GSH exhibited developmental stage-related differences with fetal being higher than neonatal or adult. The present observations may have physiologic relevance, in that decreases in pulmonary GSH levels after birth may contribute to decreases in plasma PGE2 levels by decreasing pulmonary PGE2 synthesis, thereby contributing to closure of the ductus arteriosus; conversely, increased GSH levels associated with hyperoxia may contribute to persistence of ductal patency. Formation of 6-keto-PGF1 alpha and of TXB2 (the stable metabolites of prostacyclin and TXA2) was decreased when PGE2 formation was increased by GSH activation of PGE2 isomerase in fractions isolated from all three developmental stages. A similar pattern of product formation was observed when AA was employed as substrate. These data suggest the possibility that changes in GSH concentration may modulate eicosanoid formation in cells that contain GSH-dependent PGE2 isomerase, as well as either or both prostacyclin or thromboxane synthase(s).

Cyclic GMP-dependent protein kinase expression in coronary arterial smooth muscle in response to balloon catheter injury.

Arterial smooth muscle cells undergo phenotypic and proliferative changes in response to balloon catheter injury. Nitric oxide (NO) and cGMP have been implicated in the inhibition of vascular smooth muscle cell proliferation and phenotypic modulation in cultured-cell studies. We have examined the expression of the major cGMP receptor protein in smooth muscle, cGMP-dependent protein kinase I (PKG), in response to balloon catheter injury in the swine coronary artery. On injury, there was a transient decrease in the expression of PKG in neointimal smooth muscle cells when compared with medial smooth muscle cells. The decrease in PKG expression was observed in the population of proliferating cells expressing the extracellular matrix protein osteopontin but not in cells present in the uninjured portion of the media. Coincident with the suppression of PKG expression in neointimal cells after injury, there was a marked increase in the expression of type II NO synthase (inducible NOS [iNOS], NOS-II) in the neointimal cells. These results suggest that PKG expression is transiently reduced in response to injury in the population of coronary arterial smooth muscle cells that are actively proliferating and producing extracellular matrix proteins. The reduction in PKG expression is also correlated temporally with increases in inflammatory activity in the injured vessels as assessed by iNOS expression. Coupled with our current knowledge regarding the role of PKG in the regulation of cultured cell phenotypes, these results imply that PKG may also regulate phenotypic modulation of vascular smooth muscle cells in vivo as well.

Analysis of cardiovascular responses to PACAP-27, PACAP-38, and vasoactive intestinal polypeptide.

Responses to pituitary adenylate cyclase polypeptide (PACAP)-27, PACAP-38, and vasoactive intestinal peptide (VIP) were compared in the peripheral and pulmonary vascular beds of the cat and in the isolated perfused neonatal pig heart. Intravenous injections of PACAP-27 and PACAP-38 produced biphasic changes in systemic arterial pressure whereas iv injections of VIP caused only decreases in arterial pressure. When blood flow to the hind limb and mesenteric vascular beds was maintained constant, PACAP-27 and PACAP-38 caused dose-related biphasic changes in perfusion pressure, whereas VIP only decreased perfusion pressure. PACAP-27 was approximately threefold more potent than PACAP-38, and the pressor component of the biphasic response was blocked by alpha-adrenergic antagonists and adrenalectomy. PACAP-27, PACAP-38, and VIP produced decreases in pulmonary vascular resistance, and all three peptides had significant vasodilator activity in the isolated perfused neonatal pig heart. Although all three peptides decreased coronary vascular resistance, only PACAP-27 and PACAP-38 increased left ventricular contractility, with PACAP-27 approaching isoproterenol in potency. The results of these experiments show that PACAP-27, PACAP-38, and VIP have significant effects on vasomotor tone that depend on the vascular bed studied and the contribution of adrenal catecholamines.

Prostacyclin synthetase activity in diabetic human venous tissue.

The purpose of this study was to ascertain if alterations were present in the prostacyclin synthetase (PGI2ase) activity in diabetic human venous tissue. Saphenous veins were obtained from a group of 12 patients with (HSV-D) or without (HSV-ND) diabetes who were undergoing coronary artery bypass surgery. 14C-Labeled prostaglandin endoperoxide (PGH2) was incubated for 2 minutes with venous microsomal protein. The products were separated by thin-layer chromatography and quantified by radiochromatographic scan. PGI2ase activity was determined by the formation of 6-keto-PGF1 alpha, the stable breakdown product of prostacyclin (PGI2). Results of this study indicate the following: both HSV-ND and HSV-D specimens have active PGI2ase and are capable of forming PGI2; there is no difference between PGI2ase activity in HSV-D and HSV-ND specimens; and in diabetes mellitus, any defects in PGI2 production similar to those associated with diabetes in other investigations must reside higher in the arachidonic acid cascade.

Blockade of thromboxane responses in the airway of the cat by SQ 29,548.

The effects of SQ 29,548, a thromboxane receptor antagonist, on airway responses were investigated in paralyzed, anesthetized, mechanically ventilated cats. Intravenous injections of the thromboxane and prostaglandin precursor, arachidonic acid (AA), and the thromboxane mimic, U 46619, produced dose-related increases in transpulmonary pressure and lung resistance and decreases in dynamic compliance. After administration of SQ 29,548 (0.5 mg/kg iv), bronchoconstrictor responses to AA were reduced by approximately 50%, whereas responses to U 46619 were reduced by approximately 90%. The cyclooxygenase inhibitor, sodium meclofenamate (2.5 mg/kg iv), blocked the component of the airway response to AA remaining after treatment with SQ 29,548. The thromboxane receptor antagonist had no significant effect on bronchoconstrictor responses to prostaglandins F2 alpha, and D2, methacholine, 5-hydroxytryptamine, histamine, or BAY K 8644, an agent that promotes calcium entry. Reductions in systemic arterial pressure in response to AA were enhanced by the thromboxane receptor antagonist and abolished by meclofenamate. SQ 29,548 had no effect on terminal enzyme activity in microsomal fractions from cat lung. These data support the hypothesis that AA-induced bronchoconstriction in the cat is mediated in large part by the actions of thromboxane A2. These data also suggest that U 46619 and U 44069 stimulate the same airway receptor as thromboxane A2 and mimic the bronchomotor effects of this hormone, which has not yet been isolated as a pure substance. These data demonstrate that SQ 29,548 is a selective thromboxane receptor antagonist in the airways of the closed-chest cat and may be a useful probe for studying responses to thromboxane A2 in physiological and pathophysiological processes in the lung.

Effects of OKY 1581 on bronchoconstrictor responses to arachidonic acid and PGH2.

The influence of OKY 1581, a thromboxane synthase inhibitor, on airway responses to arachidonic acid and endoperoxide, [prostaglandin (PG) H2], were investigated in anesthetized, paralyzed, mechanically ventilated cats. Intravenous injections of arachidonic acid and PGH2 caused dose-related increases in transpulmonary pressure and lung resistance and decreases in dynamic and static compliance. OKY 1581 significantly decreased airway responses to arachidonic acid but not to PGH2. Sodium meclofenamate, a cyclooxygenase inhibitor, abolished airway responses to arachidonic acid but had no effect on airway responses to PGH2. OKY 1581 or meclofenamate has no effect on airway responses to PGF2 alpha, PGD2, or U 46619, a thromboxane mimic. In microsomal fractions from the lung, OKY 1581 inhibited thromboxane formation without decreasing prostacyclin synthesis or cyclooxygenase activity. These studies show that OKY 1581 is a selective thromboxane synthesis inhibitor in the cat lung and suggest that a substantial part of the bronchoconstrictor response to arachidonic acid is due to thromboxane A2 formation. Moreover, the present data suggest that airway responses to endogenously released and exogenous PGH2 are mediated differently and that a significant part of the response to exogenous PGH2 may be due to activation of an endoperoxide/thromboxane receptor, since responses to PGH2 are blocked by the thromboxane receptor antagonist SQ 29548.

Differential effects of PACAP and VIP on the pulmonary and hindquarters vascular beds of the cat.

Responses to pituitary adenylate cyclase-activating polypeptide (PACAP), a novel peptide derived from ovine hypothalamus with 68% sequence homology with vasoactive intestinal polypeptide (VIP), were investigated in the pulmonary and hindquarters vascular beds of the anesthetized cat under conditions of controlled blood flow. Injection of the peptide into the perfused lung lobe under elevated tone conditions produced dose-dependent decreases in lobar arterial pressure that were accompanied by biphasic changes in systemic arterial pressure characterized by an initial decrease followed by a secondary increase in pressure. When compared with other vasodilator agents in the pulmonary vascular bed, the relative order of potency was isoproterenol greater than PACAP greater than acetylcholine greater than calcitonin gene-related peptide greater than VIP. In the hindquarters vascular bed, intra-arterial injections of PACAP produced biphasic changes in hindquarters perfusion pressure characterized by initial decreases followed by secondary increases, which were accompanied by biphasic changes in systemic arterial pressure. In terms of relative vasodilator activity in the hindlimb, the order of relative potency was isoproterenol greater than acetylcholine greater than calcitonin gene-related peptide greater than VIP greater than PACAP. PACAP was the only agent that caused a secondary vasoconstrictor response in the hindlimb and produced biphasic changes in systemic arterial pressure. D-Phe2-VIP, a VIP receptor antagonist, blocked the hindquarters vasodilation in response to VIP but had no effect on responses to PACAP. The present investigation shows that PACAP produces pulmonary vasodilation, as well as dilation, and vasoconstriction in the systemic (hindlimb) vascular bed.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of alcoholism and smoking on platelet eicosanoid production in vitro.

Ethanol induces changes in eicosanoid synthesis in blood platelets and brain tissue. Cigarette smoking also causes alterations in eicosanoid formation. This preliminary report examined in vitro platelet sonicate eicosanoid production using 14C-arachidonic acid (14C-AA) and in separate experiments, 14C-PGH2, as substrates. Radiometric thin layer chromatography (TLC) was used to identify the products formed. Eicosanoid product formation in platelet sonicates collected from 28 abstinent male alcoholics were compared to those from 11 male control subjects. All but one of the alcoholics were chronic smokers and all control subjects were non-smokers. All smokers abstained from smoking for 12 h prior to the blood collection to control for any acute effects of cigarette smoke on eicosanoid production. Significant reductions in platelet sonicate production of PGD2 and PGE2 in vitro were observed in alcoholic smokers when 14C-PGH2, but not 14C-AA, was the substrate. These reductions were predicted equally well by two variables, smoking and alcoholism, using several statistical models. This is the first investigation that controlled for the acute effects of smoking and accounted for the potential effects of cigarette smoking on platelet eicosanoid production in alcoholics. Because cigarette smoking is prevalent among alcoholics, future studies on the role of eicosanoids in alcoholism should control for smoking.

Influence of SQ 29,548 on vasoconstrictor responses in the hindquarters vascular bed of the cat.

The effects of SQ 29,548, a thromboxane (TX) receptor blocking agent, on vasoconstrictor responses were investigated under conditions of controlled blood flow in the hindquarters vascular bed of the cat. Intravenous injection of SQ 29,548 at a dose of 0.1 mg/kg had no significant effect on systemic arterial pressure but caused a significant reduction in hindquarters perfusion pressure. Injection of the TXA2 mimics, U44069 and U46619, into the perfusion circuit caused dose-dependent increases in hindquarters perfusion pressure with U46619 being approximately 3 times more potent than U44069. Following the administration of SQ 29,548, pressor responses to both U44069 and U46619 were reduced significantly, and the dose-response curves for both TXA2 mimics were shifted to the right in a parallel fashion. SQ 29,548 had no significant effect on the dose-dependent increases in hindquarters perfusion pressure in response to angiotensin II or BAY K8644, a nifedipine analog which promotes calcium entry. The TXA2 receptor blocking agent had no significant effect on increases in hindquarters perfusion pressure in response to angiotensin II or BAY K8644, a nifedipine analog which promotes calcium entry. The TXA2 receptor blocking agent had no significant effect on increases in hindquarters perfusion pressure in response to sympathetic nerve stimulation or injections of norepinephrine and tyramine. These findings suggest that SQ 29,548 blocks responses to the TXA2 mimics in a competitive manner, and that this inhibitory effect is selective.(ABSTRACT TRUNCATED AT 250 WORDS)

Prostaglandin E2 formation by rat gastroduodenal tissue following intragastric acid perfusion.

Rat gastroduodenal mucosa forms prostaglandin (PG) E2. However, little is known about regional differences in PGE2 formation or the effect of gastric hydrochloric acid (HC1) perfusion on regional PGE2 formation. In this study, the rats were divided into 3 groups. Group 1 received intravenous (i.v.), 1 Ml/h, and intragastric (i.g.), 8 ml/h, perfusions of saline simultaneously for 3 h. Group 2 received saline i.v. and 0.15 N HC1 i.g., 8 ml/h. Group 3 was injected with a bolus of asprin (ASA), 60 mg/kg, followed by ASA, 40 mg/kg/h i.v., and 0.15 N HC1 i.g.. The gastric aspirates were analyzed for volume and pH. Segments of gastroduodenal tissue from the fundus, corpus, antrum, and duodenum were minced and then incubated in 1 ml of 5 mM Tris buffer, pH 8.4, for 30 sec with mixing; the incubate was assayed for PGE2 by radioimmunoassay. Intragastric HC1 decreased the pH of aspirate without producing gastric mucosal lesions. However, when combined with i.v. ASA, ulcer formation was present in all animals (p less than 0.05). PGE2 was formed by isolated tissue from four different gastroduodenal regions. The duodenum formed significantly greater amounts than the fundus, antrum, or corpus, which were similar. Intragastric HC1 produced a trend toward increased PGE2 formation (pmol PGE2/mg tissue) in the fundus, 143 +/- 36 to 237 +/- 57; corpus, 87 +/- 13 to 200 +/- 57; antrum, 157 +/- 28 to 224 +/- 65; and duodenum, 235 +/- 56 to 338 +/- 51. However, statistical significance was not reached.

Selective and complete blockade of acetylcholine-induced relaxation in rabbit aortic rings by N omega-nitro-L-arginine but not by glybenclamide.

This study addressed the possibility that acetylcholine-induced relaxation in the rabbit aorta is mediated by dual mechanisms: one N omega-nitro-L-arginine (NLA)-sensitive, the other glybenclamide-sensitive. Acetylcholine, nitroglycerin and BRL38227 (lemakalim), an activator of glybenclamide-sensitive potassium channels, were added to an organ bath containing rabbit aortic rings in a cumulative manner in the absence or presence of NLA and/or glybenclamide. NLA inhibited acetylcholine-induced relaxation and potentiated the relaxant response to nitroglycerin. BRL38227 caused a dose-dependent relaxation in rabbit aortic rings, and 30 microM glybenclamide produced essentially complete inhibition of this relaxation. Glybenclamide alone produced no inhibition of acetylcholine-induced relaxation. These results indicate that glybenclamide-sensitive potassium channels in the rabbit aorta play no role in mediating the relaxant response to acetylcholine, while NLA can produce a selective and essentially complete blockade of the relaxant response to acetylcholine in the rabbit aorta.

Nisoldipine inhibits adrenergic responses in the hindquarters vascular bed of the cat.

The effects of the calcium entry blocking agent nisoldipine on adrenergic vasoconstrictor responses were investigated in the hindquarters vascular bed of the cat under conditions of controlled blood flow. Nisoldipine dilated the hindquarters vascular bed and inhibited vasoconstrictor responses to Bay K 8644, a nifedipine analog which promotes calcium entry. During infusion of nisoldipine, vasoconstrictor responses to sympathetic nerve stimulation, norepinephrine, and tyramine were inhibited in a reversible manner. In addition to blocking responses to nerve-released and exogenous norepinephrine, the calcium entry antagonist decreased responses to methoxamine and BHT 933, alpha 1- and alpha 2-adrenoceptor agonists. Responses to methoxamine were reduced by prazosin, an alpha 1-adrenoceptor antagonist, but not by yohimbine, an alpha 2-adrenoceptor blocking agent, whereas responses to BHT 933 were decreased by yohimbine but not by prazosin. The results of these studies suggest that vasoconstrictor responses to neuronally released and exogenous norepinephrine, as well as to selective alpha 1- and alpha 2-adrenoceptor agonists, are dependent in part on an extracellular source of calcium in resistance vessels of the feline hindquarters vascular bed. The inhibitory effect of nisoldipine on vasoconstrictor responses to neuronally released norepinephrine may be important in the antihypertensive actions of calcium entry blocking agents.

Effects of porcine and rat endothelin and an analog on blood pressure in the anesthetized cat.

Arterial responses to a wide range of doses of porcine and rat endothelin and a monocyclic analog were compared in the anesthetized cat. Injections of the porcine peptide in doses of 0.01-0.1 nmol/kg i.v. decreased systemic arterial pressure in a dose-related manner, whereas doses of 0.3 and 1 nmol/kg i.v. elicited biphasic responses. The rat peptide, in doses of 0.03-1 nmol/kg i.v., also decreased arterial pressure in a dose-related fashion, whereas injection at 3 nmol/kg i.v. caused a biphasic response. With both peptides the biphasic response was characterized by an initial short-lived decrease followed by a secondary sustained increase in pressure. The monocyclic porcine analog in doses of 3-30 nmol/kg i.v. had no significant effect on arterial pressure. Both peptides increased cardiac output, and changes in peripheral vascular resistance in response to both peptides were not altered by sodium meclofenamate. These data suggest that arterial depressor responses to porcine and rat endothelin are similar and dose-dependent. However, the porcine peptide has 3-fold greater pressor activity in the cat. The lack of effect with the monocyclic porcine analog suggests that the two disulfide linkages are necessary for activity.

Effect of combination endothelial nitric oxide synthase gene therapy and sildenafil on erectile function in diabetic rats.

Erectile dysfunction associated with diabetes mellitus is caused in part by disordered endothelial smooth muscle relaxation, neuropathy, and a decrease in cavernosal nitric oxide synthase (NOS) activity. The purpose of this study was to determine whether a combination of sildenafil and adenoviral gene transfer of endothelial NOS (eNOS) could enhance the erectile response in diabetic rats. Five groups of animals were utilized: (1) age-matched control rats, (2) streptozotocin (STZ)-induced diabetic rats (60 mg/kg i.p.), (3) STZ-rats + sildenafil (2 mg/kg i.v.), (4) STZ-rats transfected with AdCMVbetagal or AdCMVeNOS, and (5) STZ-rats transfected with AdCMVeNOS +sildenafil (2 mg/kg i.v.). At 2 months after i.p. injection of STZ, groups 4 and 5 were transfected with the adenoviruses and 1-2 days after transfection, all animals underwent cavernosal nerve stimulation (CNS) to assess erectile function. Cyclic 3',5'-guanosine monophosphate (cGMP) levels were assessed in the cavernosal tissue. STZ-diabetic rats had a significant decrease in erectile function as determined by the peak intracavernosal pressure (ICP) and total ICP (area under the erectile curve; AUC) after CNS when compared to control rats. STZ-diabetic rats+AdCMVeNOS had a peak ICP and AUC, which were similar to control animals. STZ-diabetic rats administered sildenafil demonstrated a significant increase in peak ICP at the 5 and 7.5 V settings, while the AUC was significantly increased at all voltage (V) settings. The increase in both ICP and AUC of STZ-diabetic rats transfected with AdCMVeNOS at all V settings was greater than STZ-diabetic rats transfected with AdCMVbetagal. STZ-diabetic rats transfected with AdCMVeNOS and administered sildenafil had a significant increase in total ICP that was greater than eNOS gene therapy alone. Cavernosal cGMP levels were significantly decreased in STZ-diabetic rats, but were increased after transfection with AdCMVeNOS to values greater than control animals. In conclusion, overexpression of eNOS and cGMP in combination with sildenafil significantly increased both the peak ICP and total ICP to CNS in the STZ-diabetic rat, which was similar to the response observed in control rats. Moreover, the total erectile response was greater in STZ-diabetic rats receiving eNOS gene therapy plus sildenafil than STZ-rats receiving sildenafil or eNOS gene therapy alone.

Analysis of responses of allicin, a compound from garlic, in the pulmonary vascular bed of the cat and in the rat.

Allicin, diallyl disulfide-oxide, an active ingredient released from garlic is a systemic vasodilator that acts by an unknown mechanism. In the present experiments, pulmonary vascular responses to allicin (0.1-1.0 mg) were studied in the intact-chest anesthetized cat and in the isolated lung of the rat under constant flow conditions. When baseline tone in the pulmonary vascular bed of the cat was raised with U46619 (11 alpha,9 alpha-epoxymethano-9 alpha,11 beta-dideoxyprostaglandin F2 alpha), intralobar injections of allicin produced dose-related decreases in pulmonary arterial pressure without changing left atrial pressure indicating that allicin had significant vasodilator activity in the pulmonary vascular bed when tone was increased experimentally. Allicin also decreased systemic arterial pressure in a dose-related manner. In terms of relative vasodilator activity in the cat, allicin was 100-fold less potent than sodium nitroprusside and many orders of magnitude less potent than isoproterenol. In the cat, vasodilator responses to allicin were unchanged by methylene blue or N omega-nitro-L-arginine methyl ester. Allicin also significantly diminished the pulmonary pressor response to ventilatory hypoxia in the isolated perfused rat lung. These data show that allicin has significant vasodilator activity in the pulmonary vascular bed of the cat and the rat. The present data suggest that pulmonary vasodilator responses to allicin are independent of the synthesis of endothelial-derived relaxing factor or the activation of soluble guanylate cyclase.

Influence of SQ 29,548 on vasoconstrictor responses in the mesenteric vascular bed of the cat.

The effects of SQ 29,548 on vasoconstrictor responses were investigated in the feline mesenteric vascular bed. Injections of the thromboxane (TX) A2 mimics, U46619 and U44069, caused dose-related increases in mesenteric arterial perfusion pressure. After administration of SQ 29,548, 0.5 mg/kg i.v, vasoconstrictor responses to U46619 and U44069 were reduced markedly whereas responses to prostaglandin (PG) F2 alpha, angiotensin II, vasopressin and BAY K 8644, an agent which enhances calcium entry, were not altered. The duration of the TXA2 receptor blockade was greater than 2 h and SQ 29,548 had no significant effect on mesenteric vasodilator responses to PGE2, isoproterenol, nitroglycerin, acetylcholine or bradykinin. SQ 29,548, at a dose of 0.5 mg/kg i.v., significantly reduced the response to TXB2, which had modest vasoconstrictor activity in the mesenteric vascular bed. However, when the dose of SQ 29,548 was reduced to 0.05 mg/kg i.v., responses to TXB2 were not altered, whereas responses to U46619 were significantly decreased. SQ 29,548 had no significant effect on vasoconstrictor responses to norepinephrine or to sympathetic nerve stimulation. The TXA2 receptor antagonist blocked the vasoconstrictor component of the biphasic response to the PG precursor, arachidonic acid, and the endoperoxide, PGH2. The results of these studies suggest that SQ 29,548 is a specific TX receptor antagonist in the mesenteric vascular bed, that the vasoconstrictor component of the biphasic response to arachidonic acid and PGH2 is due to formation of TXA2, and that endogenously formed TXA2 does not modulate adrenergic responses in the mesenteric circulation of the cat.

Synthetic human adrenomedullin and adrenomedullin 15-52 have potent short-lived vasodilator activity in the hindlimb vascular bed of the cat.

Responses to synthetic human adrenomedullin, a novel hypotensive peptide isolated from human pheochromocytoma cells, and the carboxy terminal 15-52 amino acid fragment of adrenomedullin (ADM15-52) were investigated in the hindlimb vascular bed of the cat under constant flow conditions. Intraarterial injections of the peptides in doses of 0.01-0.3 nmol caused dose-related decreases in hindlimb perfusion pressure. When compared on a nmol basis, adrenomedullin and ADM15-52 were similar to bradykinin in vasodilator potency and were approximately 10 fold less potent than acetylcholine. The half-life of the vasodilator response to adrenomedullin and ADM15-52 ranged from 55 to 80 sec and was greater than the half-life of vasodilator responses to bradykinin in doses of 0.01-0.3 nmol and acetylcholine in doses of 0.01-0.3 nmol. The present data demonstrate that synthetic human adrenomedullin and ADM15-52 have potent but relatively short-lasting vasodilator activity in the hindlimb vascular bed of the cat. These data suggest that amino acid residues 15-52 of adrenomedullin are important for the expression of vasodilator activity in the hindlimb vascular bed of the cat.