In vivo exposure to carbon disulfide increases the contraction frequency of pregnant rat uteri through an indirect pathway

Exposure to CS2, an organic solvent, is associated with an increased rate of abnormal labor or dysmenorrhea. Contraction of quiescent uteri during pregnancy can cause preterm labor. We wish to know the effects of in vivo and in vitro exposures to CS2 on uterine contractions of mid-gestation rats. After 10-d exposure to 300 or 600 mg/kg CS2, uteri of pregnant rats were measured for contractile responses to various stimuli, such as KCl, oxytocin, carbachol or A23187, a calcium ionophore, using standard muscle bath apparatus. CS2 treatment significantly increased the contractile response to KCl, carbachol, and A23187. The increase to A23187 was the greatest. In contrast, in vitro exposure to CS2 immediately suppressed carbachol-induced contraction but did not affect spontaneous and KCl-induced contractions. Results showed the pregnant uterus of the rat is susceptible to CS2. The influence of in vivo exposure to CS2 on uterine contraction was opposite to that in vitro. The increased response of CS2-treated uteri to A23187 suggests that in vivo exposure to CS2 may sensitize contraction machinery to calcium through indirect pathways.     

Effect of nifedipine on calcium-induced contractions to potassium in the aorta and mesenteric arteries of spontaneously hypertensive and normotensive rats

Graded contractions to cumulative additions of calcium in the presence of KCl were obtained in strips of aorta and mesenteric arteries of normotensive (WKY) and spontaneously hypertensive (SHR) rats. In calcium-free medium, a maximally effective concentration of KCl produced a response that was larger in the mesenteric arteries (43-51% of control) than in the aorta (12-14% of control). The calcium channel blocker nifedipine (NFD, up to 10(-7) M) did not significantly alter these calcium-insensitive responses. The Ca2+-induced responses were inhibited by NFD, in a concentration-dependent fashion, in both vessel types of WKY and SHR rats. The aortic responses were more sensitive to inhibition by NFD than the responses of mesenteric arteries. Moreover, the aortic responses of WKY were inhibited to a greater extent than those of the SHR. The results suggest: (a) a differential calcium dependence of contractions to KCl in the vessels studied; (b) that aortic responses are dependent on NFD-sensitive voltage-sensitive Ca2+ channels to a greater extent than the responses of mesenteric arteries; and (c) that hypertension results in a decreased sensitivity of the aorta Ca2+ channels to NFD.     

miR-143 decreases prostate cancer cells proliferation and migration and enhances their sensitivity to docetaxel through suppression of KRAS

As there is increasing evidence that Rho-Rho kinase (ROCK) pathway plays an important role in the proliferation and contraction in many tissues, we investigated the contractile role of a ROCK inhibitor, fasudil, and the distribution of RhoA, RhoB, RhoC, ROCK1, and ROCK2 in the rat prostate. Twelve-week-old Sprague-Dawley rat prostate was used in this study. Rat prostatic contractile responses induced by carbachol and norepinephrine were investigated in organ bath studies without or with 10(-7), 10(-6), and 10(-5) M of a non-selective ROCK inhibitor, fasudil. Immunoblot analysis and immunohistochemical staining were performed to investigate the participation levels of RhoA, RhoB, RhoC, ROCK1, and ROCK2. The E(max) values induced by carbachol and norepinephrine were similar in the rat prostate. Fasudil significantly inhibited carbachol- or norepinephrine-induced prostatic contractions in a dose-dependent manner. Fasudil 10(-5) M reduced the initial prostatic contraction (without fasudil) to 56.7 ± 5.9% for carbachol and to 45.7 ± 12.3% for norepinephrine. Amounts of RhoA, RhoB, RhoC, ROCK1, and ROCK2 were detected by immunoblot analysis in the prostate. Immunohistochemical study revealed that RhoA, RhoB, RhoC, ROCK1, and ROCK2 were all positive in the prostatic smooth muscle, while there were some differences of distributions of Immunoreactivities between these enzymes in the prostatic glandula. Our data indicated that rat prostate contains RhoA, RhoB, RhoC, ROCK1, and ROCK2, which play an important role in the autonomic nerve-mediated contractile responses in the prostate.     

Effects of econazole on receptor-operated and depolarization-induced contractions in rat isolated aorta

In our previous study, econazole caused a decrease in serum nitrite levels in septic mice in vivo, but it enhanced the mortality rate. The aim of the study was to investigate the in vitro effects of econazole on receptor-operated and depolarization-induced contractions on endothelium-intact and -denuded rat isolated aorta. Econazole (0.1, 1 and 10 microM) significantly inhibited receptor-operated (phenylephrine, Phe) and depolarization (KCl)-induced contractions of endothelium-intact or -denuded rings in a noncompetitive and concentration-dependent manner. Removal of endothelium changed the pD'2 values only for KCl-induced responses. The pD'2 values of L-type calcium channel blocker nifedipine were significantly higher than the econazole on Phe concentration-response curves in endothelium-intact and -denuded rings. Econazole caused a biphasic response in precontracted by Phe or KCl in endothelium-intact and -denuded rings, first a transient contraction following sustained relaxation. Removal of endothelium did not affect the contractile responses induced by Phe. The contractile responses induced by 10 microM econazole in the KCl-precontracted rings were antagonized by the treatment of alpha-adrenergic receptor antagonist, phentolamine (10 microM). Deendothelization was significantly increased the IC50 values of econazole obtained from Phe- and KCl-precontractions. The relaxations induced by 10 microM econazole in endothelium-intact rings precontracted with Phe or KCl were not changed by NO synthase inhibitor, L-N(G)-nitroarginine (100 microM). The IC50 values of econazole were significantly higher than nifedipine in endothelium-intact and -denuded rings. These results suggest that econazole is a noncompetitive antagonist on alpha1-adrenoceptor-mediated and depolarization-induced contractions in rat isolated aorta by inhibiting Ca2+ entry through L-type calcium channels, and the endothelium seems to modulate vascular responses induced by this agent. The vascular effects of econazole may limit the usage of this agent in septic shock.     

The significance of extracellular Ca<Superscript>2+</Superscript> in contractile responses of chick amnion

Neurotransmitter receptors are formed during chick embryo development in the amnion, an avascular extraembryonic membrane devoid of innervation. Carbachol induces phasic and tonic contractions mediated by M₃ cholinoceptors in an amniotic membrane strip isolated from 11–14-day-old chick embryo. The carbachol effect on the amnion contractile activity was studied in normal physiological salt solution, during depolarization by K⁺, exposure to nifedipine, and in calcium-free medium. Voltage-dependent and receptor-operated Ca²⁺ channels as well as calcium from intracellular stores are involved in the contractile response to carbachol. Phasic contractions of the amnion are mainly induced by calcium ions entering through voltage-dependent calcium channels, while tonic contractions are also maintained by receptor-operated channels. Ca²⁺-activated potassium channels can serve as a negative feedback factor in regulation of the amnion contractile responses.     

The source of calcium for CCK-induced contraction of the guinea-pig gall bladder.

The sources of calcium for cholecystokinin octapeptide (CCK-OP)-induced gallbladder smooth muscle contraction are considered both extracellular and intracellular, but the relative need for intracellular calcium especially at low, physiological concentrations is not clear. To better define the calcium sources responsible for guinea-pig gallbladder contractions in vitro, we inhibited calcium influx using the calcium channel blocker, methoxyverapamil, and a calcium-free Krebs' solution. Availability and release of intracellular calcium stores were depleted by strontium substitution and ryanodine. CCK-OP was compared to bethanechol and potassium chloride (KCl). Preventing calcium influx with 10(-5) M methoxyverapamil depressed the responses to CCK-OP, bethanechol and KCl. Methoxyverapamil, however, had little effect on the time-dependent generation of tension to CCK-OP, but significantly reduced the response to bethanechol and KCl, each at ED50. The duration of the contractile response in the calcium-free Krebs' solution to CCK-OP was longer than that for bethanechol. Strontium (2.5 mM) significantly attenuated the response to CCK-OP and bethanechol, but not to KCl. Ryanodine significantly reduced contractions induced by CCK-OP but not for bethanechol, both at low dose ED25. These results indicate that contraction of the guinea-pig gallbladder induced by CCK-OP, bethanechol and KCl requires extracellular calcium influx. Further, the initiation and maintenance of contraction by CCK-OP and bethanechol necessitates calcium mobilisation from intracellular stores. CCK-OP may have a greater penchant for these calcium stores, particularly at physiological doses.     

The effect of ovariectomy on the contractile response of the rat isolated detrusor muscle and urethra

Contractile responses induced by carbachol on the detrusor muscle and by noradrenaline on the isolated urethra were compared between ovariectomized rats pretreated with estradiol (50 μg/animal s.c. twice daily for five days), untreated ovariectomized rats and intact animals. In the detrusor muscle, contractions induced by 30μM carbachol, when normalized with respect to KCl 100 mM-induced contraction, were similar for the three groups. Furthermore, contractions induced by 100 μM noradrenaline in the isolated urethra were not significatively different between groups. However, the pD₂ value for noradrenaline was greater in urethral tissue from ovariectomized rats compared with ovariectomized -estrogen treated and control rats. A similar result was found for pD₂ values for carbachol-induced contractions on the detrusor muscle. These results suggest that ovariectomy increases the sensitivity of the urinary bladder and urethra to the contractile effects of carbachol and noradrenaline, respectively and that this effect is reversed by in vivo estrogen pretreatment.     

Evaluation of the Rho A/Rho-kinase pathway in the uterus of the rat model of polycystic ovary syndrome

The aim of this study was to investigate the expression of RhoA/Rho-kinase in the uterus and the effect of Rho-kinase inhibitors on uterine contractions of dehydroepiandrosterone (DHEA) induced polycystic ovary syndrome (PCOS) rats. Forty-four female Sprague-Dawley (21 days old) rats divided into three groups: The control group (n?=?14, any procedure was not performed), vehicle group (n?=?14, 0.2?ml of sesame oil, subcutaneous injection, 20 days) and PCOS group (n?=?16, DHEA 6?mg/100?g in 0.2?ml of sesame oil, subcutaneous injection, 20 days). The myometrium thickness and uterine wet weight were assessed. The mRNA and protein expressions of Rho A, the effect of Rho-kinase inhibitors (fasudil and Y-27632) on KCl, carbachol, and PGF2α induced contractions were evaluated in the uterus. In the PCOS group, the myometrium thickness and uterine wet weight significantly increased compared to the control group and vehicle group. The mRNA expression level and the immunoreactive score of Rho A, ROCK 1, ROCK 2 were similar in all groups. In the PCOS group, KCl, carbachol, and PGF2α induced uterine contractions significantly increased compared to the control group and vehicle group. Fasudil and Y-27632 significantly inhibited KCl, carbachol, and PGF2α induced uterine contractions in all groups. In conclusion, the expression of Rho A, ROCK 1, ROCK 2 not changed although myometrium thickness, uterine wet weight and the contractile responses of uterus increased in the PCOS group. The results suggest that the Rho-kinase inhibitors effectively suppressed increased contractions in the PCOS group they might be potential therapeutic agents.     

Selective alkylation of rat urinary bladder muscarinic receptors with 4-DAMP mustard reveals a contractile function for the M2 muscarinic receptor.

Our previous data indicate that M3 muscarinic receptors mediate carbachol induced bladder contractions. The data presented here were obtained by selective alkylation of M3 receptors with 4-DAMP mustard and suggest that the M2 receptor subtype may be involved in inhibition of beta-adrenergic receptor induced relaxation, therefore, allowing recontraction. Alkylation resulted in 85% of M3 receptors and 65% of M2 receptors unable to bind radioligand as demonstrated by subtype selective immunoprecipitation. Rat bladder strips subjected to our alkylation procedure contracted submaximally, and direct carbachol contractions were inhibited by antagonists with affinities consistent with M3 receptor mediated contraction. In contrast, the affinities of antagonists for inhibition of carbachol induced recontractions following isoproterenol stimulated relaxation in the presence of 90 mM KCl, indicated a contractile function for the M2 receptor that was not observed in control strips. In conclusion, these studies demonstrate a possible role for the M2 subtype in bladder smooth muscle contraction.     

Increased reactivity in the mesenteric artery of spontaneously hypertensive rats to phorbol ester

The contraction responses of mesenteric artery from 10 week old spontaneously hypertensive rats (SHRs) and normotensive Wistar Kyoto controls (WKYs) to phorbol 12, 13 - dibutyrate (PDBu) and agents acting on the potential-operated calcium channels were compared. The vessels from the SHR were significantly more sensitive to PDBu than those from the WKY. The PDBu-induced contractions were inhibited by nifedipine. The vessels from the SHR were also more sensitive to Bay K 8644 and KCl than the WKY. Low concentrations of PDBu (1 nM) potentiated the KCl contraction significantly more in the SHR than the WKY. It is suggested that the increased reactivity to PDBu in the SHR may in part be related to changes in the activity of the potential-operated calcium channels.     

In vitro tracheal responses from mice chosen for in vivo lung cholinergic sensitivity

We selected two inbred strains of mice based on their different in vivo lung responses to intravenous acetylcholine for studies on the in vitro tracheal responses to contractile and relaxing agents. In addition, we studied the role of cyclooxygenase products on the in vitro responses. Tracheal rings were contracted with increasing concentrations of carbachol and KCl and relaxed with increasing concentrations of isoproterenol after contraction with carbachol at the concentration that produced 30, 50, and 70% of the maximal contraction (EC30, EC50, and EC70, respectively) and KCl at the EC50. Half the tracheae simultaneously underwent the same protocols after pretreatment with indomethacin (3 X 10(-6) M). Despite a severalfold difference in the maximal response to cholinergic agents in vivo, there were no significant differences between the strains in the tracheal responses to carbachol (P = 0.78) or KCl (P = 0.13) in vitro. Both strains showed inhibition of the isoproterenol relaxation by carbachol (P less than 0.0001). Multiple linear regression analysis showed that the strain that was more sensitive to carbachol in vivo was also more sensitive to isoproterenol in vitro after carbachol contraction (P = 0.014). The greater isoproterenol sensitivity of the tracheae from this strain was not present after contraction with KCl, nor were these tracheae more sensitive to relaxation with sodium nitroprusside. Indomethacin pretreatment of the tissues in vitro augmented the maximal response and the sensitivity to carbachol (P less than 0.001) and KCl (P = 0.0006), and this effect was similar in both strains. Evaluation of isoproterenol relaxation after indomethacin pretreatment was confounded by the lower concentrations of carbachol needed for contraction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelin: differential effects in vascular and nonvascular smooth muscle

Endothelin, a potent vasoconstrictor, produced concentration-dependent contractions in aorta, trachea and bladder body obtained from rat and rabbit. Contractions developed slowly, reaching maxiMum within 15-20 min. Although, in both rat and rabbit tissues, endothelin was 3- to 10-fold more potent in contracting vascular (approximate EC50, 1 nM) than nonvascular smooth muscle, rat trachea and rabbit bladder did contract in response to endothelin. Rat bladder body and rabbit trachea were the least sensitive tissues with only modest contractile responses to endothelin. To determine the role of calcium in these endothelin-induced contractions, the effects of diltiazem and nitrendipine were examined. Although diltiazem (5 x 10-5) M) or nitrendipine (10(-6) M) markedly attenuated contractions produced by KCl, neither agent significantly affected concentration response curves produced by endothelin in rabbit aorta or rat trachea. In rat aorta, nitrendipine had no effect on endothelin responses, whereas diltiazem modestly decreased the maximal contraction to endothelin. However, in rabbit bladder, both calcium channel blockers significantly decreased the maximum response to endothelin with no change in EC50. These results indicate that smooth muscle sensitivity to the contractile effects of endothelin may be both species and tissue specific.     

Cytosolic heparin inhibits muscarinic and alpha-adrenergic Ca2+ release in smooth muscle. Physiological role of inositol 1,4,5-trisphosphate in pharmacomechanical coupling

In order to test the physiological significance of inositol 1,4,5-trisphosphate (InsP3) in pharmacomechanical coupling, we have utilized two near-physiological systems, in which relatively high molecular weight solutes can be applied intracellularly and receptor coupling is retained: beta-escin permeabilization and reversible permeabilization. We showed that in smooth muscle permeabilized with beta-escin, one of the saponin esters, alpha 1-adrenergic (phenylephrine) and muscarinic (carbachol) agonists, as well as caffeine and InsP3, cause contractions mediated by Ca2+ release. These contractions were calmodulin-dependent and blocked by depletion of Ca2+ stored in the sarcoplasmic reticulum. Intracellular heparin (Mr = about 5000), a blocker of InsP3 binding to its receptor and a specific inhibitor of InsP3-induced Ca2+ release in smooth muscles, inhibited the responses to the agonists and to InsP3, but not those to caffeine, nor did it block the enhanced contractile response to cytoplasmic Ca2+ induced by agonists and by GTP gamma S. Neomycin blocked Ca2+ release induced by carbachol, but not by caffeine. In reversibly permeabilized ileum smooth muscle cells, loaded with Fura-2 acid and heparin, the intracellular heparin inhibited Ca2+ release and contractions induced by carbachol in Ca2+-free, high K+ solution. Heparin did not inhibit the high K+ contractions (with 1.2 mM Ca2+) and had no significant inhibitory effects on carbachol-induced responses in the presence of extracellular Ca2+. These results, obtained under near-physiological conditions, support the conclusion that InsP3 is the major physiological messenger of the Ca2+ release component of pharmacomechanical coupling, but not of the components mediated by Ca2+ influx or by potentiation of the contractile response to Ca2+.     

Age-related changes in urethrovesical coordination in male rats: relationship with bladder instability?

The micturition profile in conscious animals and the urethrovesical coordination in anesthetized conditions were investigated in 6- and 24-mo-old male Sprague-Dawley rats. The in vitro pharmacological responses to KCl, electrical field stimulation (EFS), carbachol, phenylephrine, and isoprenaline were determined in the isolated bladder body, the bladder neck, and urethra. A morphometric and immunohistological study has been included. During conscious cystomanometry, 63% of the aging rats but only 25% of the adult rats showed spontaneous contractions during the bladder-filling phase. In conscious aging rats, basal pressure, threshold pressure, and micturition pressure were also significantly increased. In anesthetized aging rats, a decrease in resting urethral pressure at micturition threshold and the occurrence of a significant delay in urethral relaxation during micturition were associated with an increased residual volume. In all isolated tissues, contractile response to KCl was not modified with aging, whereas age-related decreases in maximal responses to carbachol in the bladder body and to phenylephrine and carbachol in the urethra were observed. In the bladder neck only, we found a significant decrease in the amplitude of neurogenic contractions associated with fibrosis but without decrease in nerve density. These experiments show significant modifications in the voiding pattern of aging rats associated with urethral dysfunction and with regionally specific pharmacological and structural changes of the urinary tract. We propose that aging in rats is characterized by an impairment of the urethrovesical coordination, leading to bladder dysfunctions similar to those induced by bladder outlet obstruction.     

Fura 2 analysis of cytosolic calcium regulation in elutriated rat gastric parietal cells

The calcium probe, Fura 2, is used to establish and partially characterize histamine-, carbachol-, and forskolin-induced calcium transients in enriched parietal cell populations prepared by centrifugal elutriation of dispersed rat fundic mucosa cell isolates. The magnitude of the maximal carbachol response, which is blocked by atropine but not cimetidine, is nearly five times that of histamine or forskolin. Time to peak responses for carbachol, forskolin, and histamine are approximately 7, 17, and 28 sec, respectively. Carbachol-, histamine-, and forskolin-induced increases in Fura 2 fluorescence appear dependent upon extracellular calcium, since these responses are attenuated in low calcium media and blocked by EGTA in low-calcium media or by lanthanum in high- or low-calcium medium. Trifluoperazine and fenoctimine, at concentrations that inhibit secretion, have no effect on either carbachol- or histamine-induced increases in cytosolic calcium. Seven major calcium/EGTA-sensitive phosphoproteins are identified by SDS-PAGE electrophoresis of ATP 32P-labeled cell sonicates. We conclude that cytosolic calcium in enriched rat gastric parietal cell populations is regulated by secretagogue receptor-controlled calcium channels. We postulate that these channels may be controlled by cyclic AMP-dependent phosphorylation, since neither changes in cyclic AMP nor calcium alone mediate the effects of secretagogues entirely, but the interplay between these two second-messenger systems potentiates the actions of these agents. The role of cytosolic calcium as a second messenger in secretagogue action appears similar to that of cyclic AMP in that a specific cellular concentration must be reached to initiate acid secretion.     

Contractile effects of 4-aminopyridine on isolated frog rectus abdominis muscles

The contractile effects of 4-aminopyridine (4-AP) on isolated frog rectus abdominis muscles were examined, and compared with KCl-induced contractures. 4-AP (1-40 mM) caused slowly developing, concentration-dependent contractures which were not modified by (+)-tubocurarine (2.7-13.3 microM). The contractures were prolonged and very slowly relaxed (greater than 30 min) on washout. KCl-induced contractures developed more rapidly and relaxation was equally rapid, both occurring within 90 s of application and washout, respectively. KCl contractures were slightly but significantly (P less than 0.05) attenuated by (+)-tubocurarine in concentrations that blocked carbachol contractures. In calcium-free Ringer's solution, KCl (10-120 mM) responses were completely abolished, but 4-AP concentration-response curves were shifted to the right three- to four-fold. The results show that 4-AP causes contracture of the frog rectus abdominis. It is suggested that at the lower concentration employed (less than 10 mM), 4-AP increases extracellular calcium entry into the muscle, while larger concentrations produce contractures by a direct intracellular mechanism. 4-AP contractures were independent of postjunctional nicotinic cholinoceptor activation.     

Effect of verapamil on the contractions produced by high potassium and by noradrenaline in human isolated saphenous vein

The effect of a calcium channel blocker, e.g. verapamil, on the contractions produced by high potassium (K+) and noradrenalne (NA), was studied in the isolated saphenous vein in man. The aim of the present experiments was to see which of the two types of contractions was more sensitive to blockade by a calcium channel blocker, e.g. verapamil, and if verapamil had a differential effect on KCl and NA, whether this could be interpreted in terms of the presence of two calcium activation mechanisms in human saphenous vein. The results of the present investigation showed that KCl and NA contracted whereas verapamil relaxed the human saphenous vein. NA produced larger contraction (3.4 g tension) than did KCl (1.3 g tension). Lowering the calcium concentration in the external medium, from 2.5 mM to 1 mM, resulted in a reduced contraction in both NA and KCl responses, indicating dependence on influx of calcium. However, verapamil (1 microM) produced greater reduction in the KCl than NA-induced contraction, indicating that the NA contraction may involve additional mechanism, i.e. dependence on the release of calcium from intracellular Ca2+ stores. These results are in favour of the suggestion that the KCl-induced contraction was due to depolarization and voltage-dependent activation of calcium channels, whereas the NA-induced contraction was due to both depolarization and receptor-activation of the calcium channels, the latter being less sensitive to calcium channel blockers, e.g. verapamil. Thus, the KCl and NA-induced contractions in human saphenous vein may be due to two different calcium activation mechanisms; one is more sensitive (KCl) than the other (NA) to the presence of the calcium antagonist, verapamil.     

The effects of temperature on contractile mechanisms of rainbow trout (Salmo gairdneri) intestine

Experiments were designed to determine whether contractility of trout smooth muscle in vitro varied with temperature and if changes occurred at the receptor or intracellular levels. The role of calcium in contractility at various temperatures was also investigated. Isolated trout intestinal segments, approximately 2 cm in length, were suspended isometrically under 2 g tension in 10-mL organ baths containing trout Ringer's solution aerated with O2 and CO2 (95:5). Contractions of trout intestine were not statistically different at 10 and 20 degrees C for carbachol, 5-hydroxytryptamine, and KCl. However, the efficacy, but not the potency, of each agonist was decreased at 2 degrees C. Receptor-induced contractions were reduced to a greater extent at 2 degrees C and did not recover to the same extent when returned to 10 degrees C in comparison with those induced by depolarization. The calcium source for contractility was also dependent on temperature. As temperatures increased, utilization of intracellular calcium increased, as indicated by increased contractility in the absence of extracellular calcium. Thus, low temperatures decrease smooth muscle contractility by affecting receptor-mediated events rather than the intracellular contractile mechanisms. Receptor-operated agonists appear to have a higher capability of using intracellular calcium than depolarizing agents.     

Dissociation of the metabolic from the contractile response to muscarinic stimulation in the rabbit urinary bladder

The calcium dependence of contraction and NADH flurorescence was investigated in rabbit bladder stimulated with bethanechol or KCl. The absence of calcium in the bathing solution induced a rightward shift in the dose response to bethanechol for both contraction and NADH flurorescence. The contractile response was shifted to a greater degree than the fluorescence response and the maximal response to bethanechol was reduced by 80% for contraction but only 20% for NADH fluorescence. This rightward shift was also induced by the benzothiazepine calcium antagonist diltiazem (200 M) and again the contractile response was shifted significantly more than the fluorescence response. The combination of zero calcium and 200 M diltiazem virtually abolished contractions but only inhibited the NADH fluorescence by 65% at maximally effective bethanechol concentrations. Unlike the effect of diltiazem on the response to bethanechol, diltiazem (200 M) shifted both the contraction and fluorescence curves to the right equally in response to KCl stimulation. These results indicate that a metabolic response to muscarinic stimulation (decreased NADH) can occur in the absence of any observable contractile response. This metabolic response may be due to post receptor signal processing events. For KCl stimulation, the NADH response is probably secondary to and a result of the contractile response.Abbreviations ATP Adenosine Triphosphate - KCl Potassium Chloride - HPLC High Performance Liquid Chromatography - NADH reduced nicotinamide Adenine Dinucleotide - NAD Oxidized Nicotinamide Adenine Dinucleotide     

Effect of phorbol ester on contractile response of aorta from endotoxic rats

The influence of phorbol ester on the isometric contractile response of aorta from endotoxic rats was examined. In endotoxic rat aorta, the contractile responses to KCl and phorbol 12,13-dibutyrate (PDBu) were both remarkably diminished, compared to those in control rat aorta. Preincubation with PDBu augmented the aortic contractile response to KCl in both control and endotoxic rats. This augmentative effect of PDBu was significantly more pronounced in endotoxic rats than in controls. When the contractile response to 80 mM KCl reached a plateau after PDBu pretreatment, addition of 5 mM CaCl2 (final concentration) to the organ bath completely reversed the diminished contractile response of endotoxic rat aorta to the control level. These results suggest that the hyporesponsiveness of endotoxic rat aorta to KCl may be caused by decreases in both protein kinase C mediated response and calcium sensitivity of vascular smooth muscle cells.