Correlative studies on the effect of carbachol on myo-inositol trisphosphate accumulation, myosin light chain phosphorylation and contraction in sphincter smooth muscle of rabbit iris

Previously, we have reported that activation of muscarinic cholinergic receptors in the iris smooth muscle results in a rapid breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2) into 1,2-diacylglycerol and myo-inositol trisphosphate (IP3) and that the stimulated hydrolysis of this phospholipid correlates well with contraction. To determine whether or not there is a causal relationship between PIP2 breakdown and contraction, we have conducted correlative studies on the effects of carbachol (CCh) on PIP2 breakdown, measured as IP3 accumulation, myosin light chain (MLC) phosphorylation and contraction in the rabbit iris sphincter. We have also investigated the effects of time, temperature, atropine antagonism, Ca++ and C-kinase activators on the three measured responses. The data obtained can be summarized as follows: dose-response studies for IP3 accumulation, MLC phosphorylation and contraction revealed a close correlation between these responses; kinetic data on atropine antagonism showed that the three measured responses are competitively inhibited by the muscarinic antagonist; time course studies conducted at low temperature showed that the CCh-induced IP3 accumulation and MLC phosphorylation may precede contraction; time course studies on the effect of Ca++ on the three measured responses showed that IP3 release may account for the rapid phase of CCh-induced contraction and that extracellular Ca++ is essential for sustained MLC phosphorylation and the slow phase of contraction; the activity of phospholipase C, the enzyme involved in PIP2 hydrolysis, in membrane fragments from 32P-labeled sphincter muscle was found to be highly sensitive to Ca++, with half-maximal stimulation at about 1.1 microM Ca++; and phorbol 12,13-dibutyrate, but not phorbol 12-myristate 13-acetate, induced MLC phosphorylation and muscle contraction in a dose- and time-dependent manner. Phorbol 12,13-dibutyrate and ionomycin acted in a synergistic manner to elicit contraction. In conclusion, contractions by CCh in the iris sphincter may be explained on the basis of enhanced PIP2 turnover and its derived second messenger molecule(s); that there are consistent correlations, using different concentrations of CCh, atropine antagonism, time, temperature and Ca++, between the stimulated hydrolysis of PIP2, MLC phosphorylation and contraction. Finally, whereas the data presented favor the involvement of IP3 in the phasic component of the contractile response, the studies with phorbol 12,13-dibutyrate suggest that contractile regulation by 1,2-diacylglycerol, through activation of C-kinase, may be important during the tonic component of smooth muscle contraction.     

Bradykinin-induced contraction is inhibited by tiaramide, an anti-inflammatory drug, with an inhibition of increase in intracellular free calcium

The mechanism of bradykinin-induced contraction in rabbit urinary detrusor was investigated using an anti-inflammatory drug, tiaramide. The contraction as well as prostaglandin (PG) E2 release induced by bradykinin was abolished by treatment with indomethacin, indicating that the contraction was mediated by PGs. The accumulation of inositol phosphates (IP) by bradykinin was partly inhibited by treatment with indomethacin, suggesting that part of the IP accumulation was due to PGs. Although the remaining accumulation of IPs induced by bradykinin in the presence of indomethacin should elicit contraction in smooth muscle cells, indomethacin abolished bradykinin-induced contraction. The dissociation between indomethacin-induced inhibition of IP accumulation and contraction induced by bradykinin might be explained by the existence of PG-generating cells in addition to smooth muscle cells. Bradykinin stimulates phospholipase C, which leads to an increase in intracellular free Ca++, activation of phospholipase A2 and release of PGs in the PG-generating cells. The released PGs act on smooth muscle cells to elicit contractions via phospholipase C activation and Ca++ mobilization. Tiaramide inhibited the PGE2 release and contraction induced by bradykinin by reducing the arachidonic acid release from membrane phospholipid but did not have a direct effect on cyclo-oxygenase. Tiaramide reduced IP accumulation induced by bradykinin to an extent similar to indomethacin. However, tiaramide had no effect on IP accumulation induced by PGE2, although it potently inhibited the contraction induced by PGE2, which elicits contractions without affecting phospholipase A2. The rise in intracellular free Ca++ induced by PGE2 as well as bradykinin was inhibited by tiaramide.(ABSTRACT TRUNCATED AT 250 WORDS)     

M2 muscarinic receptor subtype is associated with inositol trisphosphate accumulation, myosin light chain phosphorylation and contraction in sphincter smooth muscle of rabbit iris

The relationships between occupancy of muscarinic acetylcholine receptors on iris sphincter muscle, measured by [3H]quinuclidinylbenzylate (QNB) binding, carbachol (CCh)-stimulated phosphatidylinositol 4,5-bisphosphate hydrolysis, measured as myo-inositol trisphosphate (IP3) accumulation, myosin light chain (MLC) phosphorylation and contraction were analyzed by examination of the dose-response relationships and the effects of the muscarinic antagonists, atropine and pirenzepine (PZ). CCh caused a concentration-dependent accumulation of IP3 (EC50 = 2.3 X 10(-6) M), MLC phosphorylation (EC50 = 3.8 X 10(-6) M), contraction (EC50 = 0.55 X 10(-6) M) and [3H]QNB displacement [KH (high affinity dissociation constant) = 2.9 X 10(-6) M]. The time course of atropine reversal of CCh-induced IP3 accumulation and muscle contraction revealed that the continued presence of activated muscarinic acetylcholine receptors was required to maintain IP3 production and contraction. Atropine was about 2 orders of magnitude more potent than PZ in inhibiting the CCh-induced biochemical and pharmacological responses and [3H] QNB binding, indicating the preponderance of M2 receptors in this smooth muscle. Thus, the PA2 values for atropine antagonism of CCh-stimulated IP3 accumulation, MLC phosphorylation and contraction were 9.1, 9.05 and 9.39, respectively, and for PZ antagonism were 7.12, 7.10 and 7.29, respectively. Furthermore, the KD values for atropine and PZ antagonism of [3H]QNB binding were 6.9 X 10(-10) and 1.5 X 10(-7) M, respectively. In addition, AF-DX116 (11-[(2-[(diethylamino)methyl]-1-piperidinyl) acetyl]-5,11-dihydro-6 H-pyrido[2,3-b][1,4]benzodiazepine-6-one), a M2 cardioselective antagonist, significantly inhibited the CCh-induced IP3 accumulation and muscle contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enhancement of prostaglandin output during activation of beta-1 adrenoceptors in the isolated rabbit heart

The purpose of this study was to elucidate the type of adrenoceptor that mediates the effect of adrenergic stimuli on prostaglandin (PG) synthesis in the isolated rabbit heart and to determine the relationship of the released PGs to the mechanical changes elicited by catecholamines and stimulation of the cardiac sympathetic nerves. The output of 6-keto PGF1 alpha, PGE2 and PGF2 alpha was increased by electrical stimulation of the sympathetic nerves, norepinephrine, isoproterenol, dobutamine and angiotensin II, but not by phenylephrine or isoetharine. Propranolol or atenolol, but not phentolamine or butoxamine, blocked the output of PGs elicited by adrenergic stimuli. Indomethacin prevented the increase in PG formation caused by all stimuli. Moreover, the adrenergically induced release of PGs was not related to changes in heart rate, systolic tension or vascular tone elicited by the adrenergic stimuli. These data indicate that the adrenergically induced release of PGs in the isolated rabbit heart is due to the activation of beta-1 adrenoceptors and is independent of the mechanical effects produced by the adrenergic stimuli.     

Site and mechanism of growth inhibition by prostaglandins. I. Active transport and intracellular accumulation of cyclopentenone prostaglandins, a reaction leading to growth inhibition

Cyclopentenone prostaglandins (PGs) such as PGA2 or 9-deoxy-delta 9,12-13,14-dihydro-PGD2 (delta 12-PGJ2) induce growth inhibition of various lines of cultured cells. Action sites of these PGs were studied by incubating them with L-1210 murine leukemia cells. L-1210 cells accumulated both PGs in a time-dependent manner at 37 degrees C. When the uptake was analyzed with various concentrations of delta 12-PGJ2, the Michaelis-Menten type of kinetics was obtained, and the Km and Vmax were 250 microM and 2.5 nmol/min/10(6) cells, suggesting that the uptake was a carrier-mediated active transport. Competition studies with [3H]delta 12-PGJ2 showed that PGA2 was transported by the same carrier with a similar affinity. PGs without growth inhibitory activity such as PGD2, PGE2 and PGF2 alpha were neither taken up by the cells nor interfered the uptake. Subcellular distribution studies with sucrose density gradient centrifugation showed that transported delta 12-PGJ2 was present mainly in cytoplasm and nuclei without metabolism. Accumulation of the PG was attenuated greatly by preincubation of the cells at 37 degrees C for 30 min. When the effect of delta 12-PGJ2 was examined in the control and attenuated cells, a clear correlation was observed between the accumulation of the PG and its growth inhibitory effect. These results suggested that uptake and intracellular accumulation of cyclopentenone PGs are responsible for their growth inhibitory activity.     

Prostaglandin receptors EP2, EP3, and IP mediate exudate formation in carrageenin-induced mouse pleurisy

The roles of prostaglandins (PGs) as mediators of inflammation have been extensively studied, and production of PGI2 and PGE2 at inflammatory sites has been reported. However, it has not yet been clarified which type of PG receptors has a major role in inflammatory exudation. To examine in vivo role of PG receptors in inflammatory exudation, we induced pleurisy in PG receptors (IP, EP1, EP2, EP3, or EP4) knockout mice by intrapleural injection of carrageenin. Pleural exudate accumulation in wild-type (WT) mice at 1 to 5 h, but not at 24 h, was significantly attenuated by the pretreatment with indomethacin, indicating that PGs are responsible for exudate formation at the early phase of pleurisy. Pleural exudation at 1 to 5 h in IP, EP2, or EP3 knockout mice, but not in EP1 and EP4 knockout, was significantly reduced compared with in WT mice. In the exudates, 6-keto-PGF1alpha and PGE2 were detected as the major PGs, each with its peak concentration at 3 h. In addition, involvement of bradykinin in the phenomenon was suggested by the fact that captopril, a kininase inhibitor, enhanced the exudate formation and increased the amount of 6-keto-PGF1alpha and PGE2 and that a bradykinin B2-receptor antagonist inhibited the exudate formation. In contrast, leukocyte migration into pleural cavity was not influenced by indomethacin-treatment nor by these receptor deficiencies. These results demonstrate participation of EP2 and EP3 along with IP in pleural exudate formation but not in leukocyte migration in carrageenin-induced mouse pleurisy.     

Cyclic adenosine 3',5'-monophosphate in prostaglandin-induced abortion

Cyclic AMP (cAMP) in blood samples was followed during abortions induced with an intraamniotic injection of prostaglandin F2a (PGF2a) and E2 (PGE2) in women in the second trimester. The effect of intraamniotic administration of PGE2 on tissue cAMP levels and adenyl cyclase (AC) and cyclic nucleotide phosphodiesterase (PDE) in myometrium, maternal abdominal rectus muscle, placenta, and fetal liver and leg muscle was studied in women undergoing abortion with hysterotomy. Saline was injected intraamniotically into controls. Plasma cAMP values showed inconsistent variation after injection of both types of PGs. On administration of PGE2 uterine contraction started after 3 minutes. Myometrial cAMP increased 4-fold within 10 minutes, and these values also showed inconsistent variation. Neither AC or PDE activities were affected in myometrium by PGE2.     

Influence of prostaglandins E, A and F on vasoconstrictor responses to norepinephrine, renal nerve stimulation and angiotensin in the feline kidney.

The effects of intrarenal infusion of prostaglandins (PGs) of the E, A and F series on renal vascular resistance and on vasoconstrictor responses to renal nerve stimulation (RNS), norepinephrine (NE) and angiotensin (A) were determined in the in situ feline kidney under conditions of controlled blood flow. Infusion of PGE2 (3 and 0.3 mug/min) and PGE1 (3 mug/min) resulted in a marked decrease in renal perfusion pressure and a reduction in responses to all vasoconstrictor stimuli. PGE2 (0.03 mug/min) did not alter perfusion pressure. However, responses to RNS and A but not to NE were attenuated. PGA2 (3 and 0.3 mug/min) had no significant effect on perfusion pressure. PGA1 (3 mug/min) resulted in a transient decrease in renal vascular resistance which was not maintained during the infusion period. PGA2 (3 mug/min) reduced the response to RNS at 10 and 30 cps and reduced the response to A, whereas responses to NE were not affected. PGA2 (0.3 mug/min) had no effect on responses to either of the pressor stimuli. PGA1 infusion resulted in an enhanced response to RNS at the highest stimulus frequency and decreased the response elicited by A. PGF2alpha (3 mug/min) had no significant effect on renal vascular resistance or on responses to NE and nerve stimulation. However, the response to angiotensin was decreased and responses to RNS at 10 and 30 cps were decreased 30 minutes after the PGF2alpha infusion. The present data demonstrate that, of the natural renal PGs, PGE2 and PGA2 possess the capacity to modulate the effects of the sympathetic nervous system on the feline kidney. In addition, the effects of PGE and PGA on responses to adrenergic stimuli and on vascular resistance could be separated.     

Relaxant and contractile responses to prostaglandins in premature, newborn and adult baboon cerebral arteries

Dose-dependent actions of prostaglandins (PGs) were investigated on cerebral arterial strips isolated from premature, newborn and adult baboons. PGE1 an PGE2 in low concentrations (10(-9) to 10(-7) M) elicited significant relaxation in both premature and newborn baboon cerebral arteries. Arteries from adult baboons showed slight or small relaxation in response to these PGs. PGE1 and PGE2 in higher concentrations (10(-8) to 10(-6) M) caused no contraction in premature and newborn arteries, but significant contraction in adult arteries. PGF2 alpha (10(-9) to 10(-7) M) elicited relaxations in arteries from baboons of every age group, being greater in prematures and newborns than in adults. PGF2 alpha (3 X 10(-7) to 10(-5) M) produced a slight or small contraction in prematures and newborns, respectively, whereas larger contraction was induced in the adult artery. PGI2 (prostacyclin) (10(-8) to 10(-6) M) produced dose-dependent relaxation in arteries from baboons of all age groups with no significant difference in the relaxant effect among the three age groups. Effective concentration (EC25) values for relaxant effect of PGE1 and PGE2 were much less than those of PGF2 alpha and PGI2 in premature and newborn arteries. In adult cerebral arteries, only PGF2 alpha and PGI2 were effective in causing a significant relaxation. In premature and newborn arteries, PGE1 and PGE2 were not effective in causing a significant contraction, whereas in adult arteries EC25 values for contractile effects of PGE1 and PGE2 were less than those for PGF2 alpha.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glomerular prostaglandins: synthesis, mechanism of action and interaction with ACE inhibitors]

Prostaglandins (PG) play an important role in the regulation of the renal blood flow and glomerular filtration rate. This study was designed to examine PG synthesis in the presence and absence of the ACE inhibitor captopril, PG binding to specific receptors and the ability of PG to stimulate cAMP accumulation in isolated glomeruli. Glomeruli were isolated from rat kidneys by a passive mechanical sieving technique. PG synthesis was determined by RTLC and RIA. The main eicosanoids synthesized by glomeruli were PGF2 alpha, thromboxane (TX) A2 (measured as TXB2), PGI2 (measured as 6-keto-PGF1 alpha) and PGE2. Binding experiments were performed with PGE1, PGE2 and the PGI2 analogue iloprost. Scatchard analysis revealed that the specific binding was highest for PGE1, followed by iloprost and PGE2. Adenylate cyclase was preferentially stimulated by PGE1 and PGE2, and to a lesser extent by PGI2, whereas PGF2 alpha had almost no effect. Captopril reduced mainly TXB2 concentrations. Glomerular TXB2 reduction, therefore, seems to be an additional hypotensive effect of captopril medication.     

Location of enkephalinase and functional effects of [Leu5]enkephalin and inhibition of enkephalinase in the feline main pancreatic and bile duct sphincters.

1. Morphological studies have demonstrated enkephalinergic nerve fibres in proximity to the sphincter of Oddi, and opiates are known to contract this sphincter. In this study, the flow resistances in the common bile duct and main pancreatic duct sphincters were studied simultaneously in anaesthetized cats using a perfusion technique. 2. Naloxone did not affect the activity of these sphincters under basal conditions, indicating that there is no basal enkephalinergic tone. 3. The response to [Leu5]enkephalin (0.015-15 micrograms/kg), morphine (1 mg/kg) and ketamine (10 mg/kg) was a naloxone-sensitive increased activity in the sphincters with a raised frequency of phasic contractions. The threshold dose for an effect of [Leu5]enkephalin on the sphincter of Oddi was 0.015 microgram/kg and a maximal response was observed at 0.75 microgram/kg. There were no differences in the response of the main pancreatic duct sphincter and the bile duct sphincter to the different drugs. 4. Immunoautoradiographic studies demonstrated enkephalinase in the sphincter++ of Oddi. 5. Acetorphan (3 mg/kg intravenously), which inhibits endogenous enkephalinase both in the peripheral and the central nervous system when administered parenterally, caused a naloxone-sensitive contraction, whereas thiorphan (3-20 mg/kg), an enkephalinase inhibitor that does not easily penetrate the blood-brain barrier, had no effect on the sphincter of Oddi. 6. These results show that endogenous and exogenous opiates influence the function of the feline sphincter of Oddi and that enkephalins may be involved in the physiological control of this sphincter, although not under basal conditions.     

Endothelium-dependent and independent responses to prostaglandin H2 and arachidonic acid in isolated dog cerebral arteries

The addition of prostaglandin (PG) H2 produced a transient contraction followed by a relaxation in helical strips of dog cerebral arteries partially contracted with PGF2 alpha or K+. The contraction was abolished by removal of endothelium, and the relaxation was potentiated. Relaxation induced by PGI2 was not influenced by endothelium denudation. The PGH2-induced contraction in strips with intact endothelium was not influenced by OKY-046, a thromboxane A2 synthesis inhibitor, but was abolished by treatment with ONO3708, an antagonist of vasoconstrictor PGs, whereas the relaxation was inhibited by tranylcypromine or diphloretin phosphate, a nonselective PG antagonist. Contraction induced by arachidonic acid (AA) was reversed to relaxation by removal of endothelium or treatment with ONO3708. Treatment with indomethacin attenuated the AA-induced contraction in the intact strips and also the relaxation in the strips treated with ONO3708 or denuded of endothelium. It may be concluded that vasoconstrictor PGs are synthesized from PGH2 or AA mainly in endothelium, and the production of PGI2 from PGH2 is not dependent on endothelium. Thromboxane A2 in concentrations sufficient to elicit significant contractions does not appear to be liberated from the cerebroarterial wall stimulated by PGH2.     

Differences in responsiveness of intrapulmonary artery and vein to arachidonic acid: mechanism of arterial relaxation involves cyclic guanosine 3':5'-monophosphate and cyclic adenosine 3':5'-monophosphate

The objective of this study was to examine the relationship between responses of bovine intrapulmonary artery and vein to arachidonic acid and cyclic nucleotide levels in order to better understand the mechanism of relaxation elicited by arachidonic acid and acetylcholine. Arachidonic acid relaxed phenylephrine-precontracted arterial rings and elevated both cyclic GMP and cyclic AMP levels in arteries with intact endothelium. In contrast, endothelium-damaged arterial rings contracted to arachidonic acid without demonstrating significant changes in cyclic nucleotide levels. Indomethacin partially inhibited endothelium-dependent relaxation and abolished cyclic AMP accumulation whereas methylene blue, a guanylate cyclase inhibitor, partially inhibited relaxation and abolished cyclic GMP accumulation in response to arachidonic acid. All vessel responses were blocked by a combination of the two inhibitors. Prostaglandin (PG) I2 relaxed arterial rings and elevated cyclic AMP levels whereas PGE2 and PGF2 alpha caused contraction, suggesting that the indomethacin-sensitive component of arachidonic acid-elicited relaxation is due to PGI2 formation and cyclic AMP accumulation. The methylene blue-sensitive component is attributed to an endothelium-dependent but cyclooxygenase-independent generation of a substance causing cyclic GMP accumulation. Intrapulmonary veins contracted to arachidonic acid with no changes in cyclic nucleotide levels and PGI2 was without effect. Homogenates of intrapulmonary artery and vein formed 6-keto-PGF1 alpha, PGF2 alpha and PGE2 from [14C]arachidonic acid, which was inhibited by indomethacin. Thus, bovine intrapulmonary vein may not possess receptors for PGI2. The failure of endothelium-intact vein to relax to acetylcholine may be related to the lack of a relaxant effect by arachidonic acid, perhaps attributed to the absence of generation of an endothelium-derived relaxing factor.     

Prostaglandins in the kidney: developments since Y2K

There are five major PGs (prostaglandins/prostanoids) produced from arachidonic acid via the COX (cyclo-oxygenase) pathway: PGE(2), PGI(2) (prostacyclin), PGD(2), PGF(2alpha) and TXA(2) (thromboxane A(2)). They exert many biological effects through specific G-protein-coupled membrane receptors, namely EP (PGE(2) receptor), IP (PGI(2) receptor), DP (PGD(2) receptor), FP (PGF(2alpha) receptor) and TP (TXA(2) receptor) respectively. PGs are implicated in physiological and pathological processes in all major organ systems, including cardiovascular function, gastrointestinal responses, reproductive processes, renal effects etc. This review highlights recent insights into the role of each prostanoid in regulating various aspects of renal function, including haemodynamics, renin secretion, growth responses, tubular transport processes and cell fate. A thorough review of the literature since Y2K (year 2000) is provided, with a general overview of PGs and their synthesis enzymes, and then specific considerations of each PG/prostanoid receptor system in the kidney.     

Endothelin-1 stimulates contraction of rat glomerular mesangial cells and potentiates beta-adrenergic-mediated cyclic adenosine monophosphate accumulation.

The newly isolated peptide, endothelin-1 (ET-1), is a potent pressor agent that reduces GFR and the glomerular ultrafiltration coefficient. Recent evidence demonstrates that ET-1 mobilizes intracellular Ca2+ [( Ca2+]i) in glomerular mesangial cells by activating the phosphoinositide cascade. The present experiments were designed to examine whether ET-1 stimulates mesangial cell contraction and regulates the synthesis of PGE2 and cAMP, which dampen vasoconstrictor-induced mesangial contraction. ET-1 (greater than or equal to 1 nM) reduced the cross-sectional area of rat mesangial cells cultured on three-dimensional gels of collagen type I. ET-1 also caused complex rearrangements of F-actin microfilaments consistent with a motile response. Contraction in response to ET-1 occurred only at concentrations that activate phospholipase C, and contraction was unaffected by blockade of dihydropyridine-sensitive Ca2+ channels. Elevation of [Ca2+]i with ionomycin, to equivalent concentrations of [Ca2+]i achieved with ET-1, also reduced mesangial cell cross-sectional area. ET-1 (0.1 microM) also evoked [3H]arachidonate release and a fivefold increase in PGE2 synthesis as well as increased synthesis of PGF2 alpha and small changes of TXB2. ET-1 caused a minor increase in intracellular cAMP accumulation only in the presence of 3-isobutyl-1-methylxanthine. ET-1 also amplified cAMP production in response to isoproterenol. TPA and ionomycin, alone and in combination, failed to mimic the potentiating effect of ET-1; however, indomethacin blocked ET-1-induced potentiation of isoproterenol-stimulated cAMP, which was restored by addition of exogenous 10 nM PGE2. Thus the present data demonstrate that ET-1 stimulates mesangial cell contraction via pharmacomechanical coupling and activates phospholipase A2 to produce PGE2, PGF2 alpha, and TXB2. ET-1 also amplified beta adrenergic-stimulated cAMP accumulation by a PGE2-dependent mechanism.     

Cytochrome P450 metabolites of arachidonic acid but not cyclooxygenase-2 metabolites contribute to the pulmonary vascular hyporeactivity in rats with acute Pseudomonas pneumonia

We have previously demonstrated depressed vascular contractility in intralobar pulmonary artery (PA) rings isolated from rats with acute Pseudomonas pneumonia. Here we describe the role of arachidonic acid (AA) metabolites in the regulation of pulmonary vascular tone in inflammation. Pneumonia was induced by intratracheal injection of P. aeruginosa organisms. Rats were sacrificed 44 h later. EETs and 20-HETE were formed at significantly lower rates in pneumonia compared with control lung microsomes. Vasoactive effects of CYP metabolites (5,6-EET, 8,9-EET, 11,12-EET, 14,15-EET, and 20-HETE) on small PA rings from control or pneumonia rats were assessed in vitro. All four EETs and 20-HETE were more potent PA vasoconstrictors than KCl or phenylephrine (PE). However, this potency was attenuated in PA rings from pneumonia lungs compared with control. In contrast, pneumonia had no effect on COX activity [total pulmonary prostaglandin (PG), PGE(2), and 6-keto-PGF(1 alpha)]. In vitro vascular contractility to KCl, PE, or PGF(2 alpha) was assessed in small PA rings from control and pneumonia rats in the presence and absence of the COX-2 inhibitor NS-398 (10 microM). NS-398 did not reverse the attenuated contractile responses to KCl, PE, or PGF(2 alpha) in pneumonia rats. Nitrite/nitrate levels, inducible nitric-oxide synthase and heme oxygenase activities were all significantly elevated in pneumonia lungs. In conclusion, vasodilator PGs produced by COX-2 do not contribute to the depressed PA contractility in this model of pneumonia. Depressed pulmonary production and vasoconstrictor effects of CYP metabolites of AA (possibly due to increased NO and/or carbon monoxide) indicate a potential role for these vasoactive metabolites in this model of acute pneumonia.     

Role of phosphoinositide turnover and cyclic AMP accumulation in prostaglandin-stimulated noradrenaline release from cultured adrenal chromaffin cells

Prostaglandins (PGs) E1, E2 and F2 alpha stimulated release of noradrenaline from chromaffin cells; the most potent was PGF2 alpha with an EC50 of about 0.1 microM. The rank order of potency for release, and the EC50 for each PG, was the same as that for stimulation of (poly)phosphoinositide turnover. PGE1-stimulated release was dependent on extracellular calcium and sensitive to dihydropyridine calcium channel agonists and antagonists at 1 microM, but unlike release stimulated by 50 mM extracellular potassium was not sensitive to verapamil or diltiazem at 10 microM. The PGs also enhanced the turnover of inositol phospholipids, but the PGE1-stimulated formation of inositol phosphates was small compared to that produced by bradykinin, which stimulates a similar degree of release. Unlike release, the stimulation of inositol phosphate formation by PGs was not dependent on the addition of calcium to the medium. In down-regulation experiments, involving 2 hr preincubation with 30 microM PGE1, release in response to the three PGs was attenuated, whereas the release response to bradykinin and nicotine was unaffected. However, the stimulation of (poly)phosphoinositide turnover by PG was not down-regulated by prior exposure to PGE1. This dissociation of the inositol phosphate response suggests that release in response to PGs is not downstream of stimulation of inositol phospholipid hydrolysis. A further series of experiments is reported which shows that release is not a consequence of increased rate of cyclic AMP synthesis. It seems likely that PG stimulation of noradrenaline release is a result of calcium entry through dihydropyridine-sensitive channels by a mechanism independent of these two second messenger systems.     

Increase of cyclic AMP concentrations in cultured vascular smooth muscle cells by vasoactive peptide hormones. Role of endogenous prostaglandins

We have evaluated the hypothesis that vasoactive hormones increase cellular cyclic AMP (cAMP) levels in cultured vascular smooth muscle cells from rat mesenteric arteries by stimulating endogenous prostaglandin (PG) synthesis. Vasopressin and angiotensin II, which were shown previously to provoke the synthesis of PGs in cultured vascular smooth muscle cells, increased cellular cAMP concentrations by about 2-fold, whereas a peptide analog of vasopressin, 1-desamino-8-D-arginine vasopressin, mostly lacking vasopressin's ability to elicit PG synthesis, was ineffective. Two other chemically dissimilar effectors that provoked the synthesis of PGs in cultured vascular smooth muscle cells, namely arachidonate and ionophore A23187, also increased cellular cAMP levels. The increase of cAMP by vasopressin and angiotensin II was transient, reaching a maximum at 1 to 2 min of incubation, followed by a decline to basal levels. Acetylsalicylic acid, a specific inhibitor of PG synthesis, completely prevented vasopressin- and arachidonate-evoked increases of cAMP but did not affect basal cAMP concentrations. Exogenous prostacyclin and prostaglandin E2 dose-dependently increased cAMP concentrations although prostacyclin was more effective than prostaglandin E2. The ability of exogenous prostacyclin to evoke cAMP increases was not inhibited by acetylsalicylic acid. The results support the hypothesis that the stimulation of endogenous PG synthesis by vasoactive hormones in turn modulates cellular cAMP levels in cultured vascular smooth muscle cells from rat mesenteric arteries.     

Prostacyclin modulates cholesteryl ester hydrolytic activity by its effect on cyclic adenosine monophosphate in rabbit aortic smooth muscle cells.

We tested the hypothesis that prostacyclin (PGI2), 6-keto-prostaglandinF1 alpha(6-keto-PGF1 alpha), and several E series prostaglandins (PG) may affect the activity of cholesteryl ester (CE) hydrolase since our previous experiments indicated that smooth muscle cells (SMC) in neointima of injured rabbit aorta (a) acquire the capacity to produce PGI2 and (b) have increased lysosomal CE hydrolytic (acid cholesteryl ester hydrolase [ACEH])activity. Using cultured SMC from rabbit thoracic aorta, we demonstrated that PGI2, 6-keto-PGF1 alpha, and 6-keto-PGE1 enhanced ACEH activity fourfold. No significant effects on ACEH activity were observed with PGE1 or PGE2. Preincubation of SMC with an inhibitor of adenylate cyclase activity (dideoxyadenosine) abolished the effect of these PG on CE hydrolytic activity. Addition of dibutyryl cAMP to these SMC significantly increased ACEH activity. Although concentrations of PGI2 used significantly increased cAMP levels, proliferation of these SMC was not observed. In related experiments, we determined if the addition of PGI2, 6-keto-PGF1 alpha, or 6-keto-PGE1 to cultured aortic SMC would enhance the egress of unesterified cholesterol and CE from these SMC. A significant loss of total cholesterol from PG-treated SMC was observed at the end of 14 d. Results suggest that increased synthesis of PGI2 by neointimal SMC in the injured aortic wall may, at least in part, explain the changes in CE catabolism and accumulation following injury. These PG may also be important in CE metabolism and accumulation in human arteries.     

Ca2+-independent, inhibitory effects of cyclic adenosine 5'-monophosphate on Ca2+ regulation of phosphoinositide 3-kinase C2alpha, Rho, and myosin phosphatase in vascular smooth muscle

We have recently demonstrated in vascular smooth muscle (VSM) that membrane depolarization by high KCl induces Ca(2+)-dependent Rho activation and myosin phosphatase (MLCP) inhibition (Ca(2+)-induced Ca(2+)-sensitization) through the mechanisms involving phosphorylation of myosin-targeting protein 1 (MYPT1) and 17-kDa protein kinase C (PKC)-potentiated inhibitory protein of PP1 (CPI-17). In the present study, we investigated whether and how cAMP affected Ca(2+)-dependent MLCP inhibition by examining the effects of forskolin, cell-permeable dibutyryl cAMP (dbcAMP), and isoproterenol. Forskolin, but not its inactive analog 1,9-dideoxyforskolin, inhibited KCl-induced contraction and the 20-kDa myosin light chain (MLC) phosphorylation without inhibiting Ca(2+) mobilization in rabbit aortic VSM. dbcAMP mimicked these forskolin effects. We recently suggested that Ca(2+)-mediated Rho activation is dependent on class II alpha-isoform of phosphoinositide 3-kinase (PI3K-C2alpha). Forskolin inhibited KCl-induced stimulation of PI3K-C2alpha activity. KCl-induced membrane depolarization stimulated Rho in a manner dependent on a PI3K but not PKC and stimulated phosphorylation of MYPT1 at Thr(850) and CPI-17 at Thr(38) in manners dependent on both PI3K and Rho kinase, but not PKC. Forskolin, dbcAMP, and isoproterenol inhibited KCl-induced Rho activation and phosphorylation of MYPT1 and CPI-17. Consistent with these data, forskolin, isoproterenol, a PI3K inhibitor, or a Rho kinase inhibitor, but not a PKC inhibitor, abolished KCl-induced diphosphorylation of MLC. These observations indicate that cAMP inhibits Ca(2+)-mediated activation of the MLCP-regulating signaling pathway comprising PI3K-C2alpha, Rho, and Rho kinase in a manner independent of Ca(2+) and point to the novel mechanism of the cAMP actions in the regulation of vascular smooth muscle contraction.