KCl-induced Tension Research Articles

Influence of conditioned media from glial cell cultures on contractility of uterine in rats of different ages

Background. The physiological regulation of the uterine contractile activity changes with age, which leads to an increased number of prolonged labor and emergency caesarean sections in women giving birth at the age of 35+. One of the modern approaches to correct the function of the reproductive system is the use of from cell cultures. CM from glial cell culture contains neurotrophic factors that play an important role in maintaining the contractile function of the uterus. Current cell culture technologies include cryopreservation.Objective: to research experimentally the effect of CM obtained from intact and cryopreserved cultures of glial cells on the contractile activity of the uterus in rats of different reproductive ages.Materials and methods. The monolayer cell culture was obtained from the dorsal root ganglia of neonatal piglets and cryopreserved in the presence of cryoprotectant dimethyl sulfoxide. CM from native and cryopreserved cultures were collected for 28 days, after which fractions with a molecular weight of < 30 kDa were obtained from them by ultrafiltration. Rats at the age of 6 and 14 months, which corresponds to reproductive age and late reproductive age (LRA), were intraperitoneally injected with 0.2 ml of media from intact (ICM) or cryopreserved (CCM) cultures for 9 days. On the 30th – 32nd day after the end of the administration of CM animals were slaughtered and the uterine contractile activity was determined by the organ bath method, the relative area of myometrium and density of myocytes by histological method, the average area of labeling with specific antibodies to smooth muscle actin by immunohistochemical method. The statistical significance of differences was assessed by the Mann–Whitney test.Results. It was found that spontaneous, OT-, and KCl-induced tension of isometric contraction of the uterus in intact LRA rats decreased by 19, 20, and 14%, respectively, compared with intact reproductive aged animals. After the introduction of ICM and CCM in LRA animals, normalization of isometric contraction parameters was observed. This effect was realized against the background of an increase in the area of the myometrium, the density of myocytes, and actin expression.Conclusions. Intra-abdominal administration of CM from glial cell culture increases the uterine contractile activity in LRA rats. This effect is realized by increasing the relative area of the myometrium, the density of myocytes, and the area of expression of smooth muscle actin. The effect of media from intact and cryopreserved cultures on the contractile activity of the uterus was similar, which makes it possible to use low-temperature culture storage technologies to obtain CM without losing its biological effect.

The Flavone Luteolin, an Endocrine Disruptor, Relaxed Male Guinea Pig Gallbladder Strips.

BackgroundLuteolin (3',4',5,7-tetrahydroxyflavone) is a flavone with a yellow crystalline appearance present in numerous plants such as broccoli, green chili, and carrot. Luteolin is considered to be an endocrine disruptor with potent estrogen agonist activity and potent progesterone antagonist activity. Luteolin has effects on smooth muscle. Luteolin relaxed guinea pig trachea smooth muscle as it inhibited both phosphodiesterase and reduced intracellular Ca2+. Luteolin also caused vasorelaxation in rat thoracic aorta smooth muscle by inhibiting intracellular Ca2+ release, inhibition of sarcolemmal Ca2+ channels, and activation of K+ channels. Luteolin or its glycosides from artichoke extracts may have an ameliorating effect on irritable bowel syndrome. The purpose of this study was to determine if luteolin had an effect on gallbladder motility.MethodsAn in vitro pharmacologic technique was utilized. Either cholecystokinin octapeptide (CCK) or KCl were used to induce tension in male guinea pig gallbladder strips maintained in Sawyer-Bartlestone chambers. Luteolin relaxed either the CCK- or KCl-induced tension in a concentration dependent manner. Various blockers were added to the chambers to determine which second messenger system(s) mediated the observed relaxation. Paired t-tests were used for statistical analysis. Differences between mean values of P < 0.05 were considered significant.ResultsTreatment of the gallbladder strips with luteolin prior to either KCl or CCK significantly (P < 0.001) decreased the amount of either KCl- or cholecystokinin-induced tension. The 2-aminoethoxydiphenylborane was used to ascertain if the release of intracellular Ca2+ mediated the luteolin-induced relaxation. It significantly (P < 0.001) decreased the amount of luteolin-induced relaxation. To ascertain if PKA mediated the luteolin-induced relaxation, PKA inhibitor 14-22 amide myristolated was used. It significantly (P < 0.01) reduced the amount of luteolin-induced relaxation. Neither KT5823, NG-methyl-L-arginine acetate salt, genistein, tetraethylammonium, nor fulvestrant had a significant effect. To ascertain if PKC mediated the luteolin-induced relaxation, the PKC inhibitors bisindolymaleimide IV and chelerythrine Cl- were used together. They had no significant effect.ConclusionsLuteolin relaxed cholecystokinin- or KCl-induced tension by blocking extracellular Ca2+ entry as well as intracellular Ca2+ release. In addition, the actions of PKA are also involved in mediating the luteolin effect.

Prostaglandin E2 Receptor EP4 Inhibition Contracts Rat Ductus Arteriosus.

Patent ductus arteriosus (PDA) is common in premature infants. Cyclooxygenase inhibitors such as indomethacin, which inhibit prostaglandin E2(PGE2) synthesis, are currently the sole treatments for patients with PDA. Their efficacy are, however, frequently limited, and adverse effects are problematic. Because the PGE2-specific receptor EP4 selectively expresses in rat ductus arteriosus (DA), it is hypothesized that EP4 inhibition would promote DA closure with fewer side-effects.Methods and Results:A new chemical compound EP4 antagonist, RQ-15986 (renamed from CJ-042794), was used. Whether RQ-15986 selectively contracted the DA was examined by measuring the isometric tension of rat DA ex vivo at embryonic day 19 (e19) and e21. RQ-15986 at a dose of 10-6mol/L increased the isometric tension of the DA up to 44.8±6.2% and 69.1±12.9% to the maximal KCl-induced tension at e19 and e21 respectively. The effect of RQ-15986 on rat DA in vivo was also tested by using a rapid whole-body freezing method. RQ-15986 inhibited PGE1-induced DA dilatation in neonatal rats. Furthermore, RQ-15986 contracted the DA in a dose-dependent manner, and the constriction was greater at e21 than at e19. Moreover, RQ-15986 did not contract the aorta or the marginal artery of the colon. EP4 inhibition contracts rat DA with fewer side-effects. EP4 inhibition is a promising alternative strategy to treat patients with PDA.

Quercetin relaxes guinea pig gallbladder strips

Quercetin, a phytoestrogen and flavonoid, relaxes intestinal and vascular smooth muscle. The purpose of this study was to determine if quercetin had an effect on gallbladder smooth muscle. An in vitro technique was used to determine the effects of quercetin on gallbladder strips and which system(s) mediated the relaxation. Paired t tests were used; differences between means of P < .05 were considered significant. Adding quercetin before cholecystokinin or KCl produced a significant (P < .001) decrease in the amount of tension (0.80 ± 0.04 vs 0.48 ± 0.04 g cholecystokinin octapeptide and 0.8 ± 0.06 vs 0.54 ± 0.05 g KCl, respectively). When the protein kinase C (PKC) inhibitors bisindolymaleimide IV and chelerythrine Cl− were simultaneously, a significant (P < .001) reduction in the quercetin-induced relaxation (45.7% ± 4.3% vs 27.6% ± 3.4%) was observed. To determine if protein kinase A (PKA) mediated the quercetin-induced relaxation, PKA inhibitor 14-22 amide myristolated was used. It significantly (P < .05) decreased the amount (40.4% ± 3.7% vs 34.5% ± 3.3%) of quercetin-induced relaxation. The use of 2-APB also significantly (P < .001) reduced the amount of quercetin-induced relaxation (51.2% ± 3.5% vs 14.8% ± 3.6%). l-NG-methyl-l-arginine acetate salt, a nitric oxide synthase inhibitor, significantly (P < 001) decreased the quercetin-induced relaxation (45.7% ± 4.2% vs 35.2% ± 3.6%). KT5823, a PKC inhibitor, had no effect on the quercetin-induced relaxation. Quercetin blocked extracellular Ca2+ entry which affected downstream events such as activation of PKC, PKA, intracellular Ca2+ release, and activation of nitric oxide synthase. Quercetin relaxed cholecystokinin octapeptide and KCl-induced tension in a concentration dependent manner. Thus quercetin-induced relaxation was mediated by multiple signaling pathways.

The Resveratrol-induced Relaxation of Cholecystokinin Octapeptide- or KCl-induced Tension in Male Guinea Pig Gallbladder Strips Is Mediated Through L-type Ca2+Channels.

Background/AimsResveratrol (3,5,4′-trihydroxystilbene) is a polyphenolic compound (stilbene) and a phytoalexin. The purpose of this study was to determine the mechanism which mediated the resveratrol-induced relaxation of cholecystokinin octapeptide- or KCl-induced tension in male guinea pig gallbladder strips.MethodsGallbladder strips were prepared and suspended in in vitro chambers filled with Krebs-Henseleit solution. The strips were attached to force displacement transducers, and the changes in tension were recorded on a polygraph. All reagents were added directly into the chambers.ResultsTo determine if intracellular Ca2+ release mediated the resveratrol-induced relaxation of cholecystokinin octapeptide-induced tension, 2-aminoethoxydiphenylborane (2-APB) was used. 2-APB significantly (P < 0.01) decreased the amount of RSVL-induced relaxation. To determine if protein kinase A (PKA) mediated the resveratrol-induced relaxation, PKA inhibitor 14-22 amide myristolated (PKA-IM) was used. PKA-IM had no effect on resveratrol-induced relaxation. Neither KT5823, NG-methyl-L-arginine acetate salt, a nitric oxide synthase inhibitor, nor fulvestrant had a significant effect on the amount of resveratrol-induced relaxation. Genistein, a protein tyrosine kinase inhibitor, significantly (P < 0.01) increased the RSVL-induced relaxation. To determine if protein kinase C mediated the RSVL-induced relaxation, the protein kinase C inhibitors bisindolymaleimide IV and chelerythrine Cl- were used together, and a significant (P < 0.05) increase in resveratrol-induced relaxation was observed. The pretreatment of the strips with resveratrol significantly (P < 0.001) decreased the amount of KCl- and cholecystokinin octapep-tide-induced tension.ConclusionsResveratrol-induced relaxation is mediated by its effects on L-type Ca2+ channels and intracellular Ca2+ release.

Curcumin Relaxes Precontracted Guinea Pig Gallbladder Strips via Multiple Signaling Pathways.

BackgroundCurcumin (diferuloymethane) is the active ingredient of the dietary spice turmeric. Curcumin modulates various signalling molecules, including inflammatory agents, transcription factors, protein kinases and cell cycle regulatory proteins. The purpose of this study was to determine if curcumin had an effect on gallbladder motility.MethodsA pharmacologic in vitro technique was used. Since curcumin relaxed both cholecystokinin octapeptide- (CCK) and KCl-induced tension of guinea pig gallbladder strips in a concentration dependent manner, an in vitro technique was used to determine which second messenger system(s) mediated the observed relaxation. Paired t-tests, t-tests and analysis of variance were used for statistical analysis. Differences between mean values of P < 0.05 were considered significant.ResultsTo determine if protein kinase A (PKA) mediated the curcumin-induced relaxation, PKA inhibitor 14-22 amide myristolated (PKA-IM) was used. PKA-IM had no significant effect on the amount of curcumin-induced relaxation. When the protein kinase C (PKC) inhibitors bisindolymaleimide IV and chelerythrine Cl- were used together, a significant (P < 0.01) reduction in the curcumin-induced relaxation was observed. The use of tetraethylammonium chloride (TEA) caused a significant (P < 0.01) decrease in the amount of curcumin-induced relaxation. Adding curcumin prior to the KCl caused a significant (P < 0.001) decrease in the amount of KCl-induced tension.ConclusionsThe results suggested that the curcumin-induced relaxation is mediated by multiple signaling pathways including the PKC second messenger system, inhibiting extracellular Ca2+ entry and K+ channels.

The flavonoid chrysin, an endocrine disrupter, relaxes cholecystokinin- and KCl-induced tension in male guinea pig gallbladder strips through multiple signaling pathways

The bioflavonoids have effects on vascular smooth muscle and gastrointestinal smooth muscle. The flavone and phytoestrogen, chrysin, has been shown to have a vasorelaxant effect on resistance blood vessels. This effect was mediated by nitric oxide (NO). Chrysin inhibited aromatase/estrogen biosynthesis in postmenopausal women. The purpose of this study was to determine if chrysin had an effect on cholecystokinin- or KCl-induced tension in male guinea pig gallbladder strips. In addition, the second messenger(s) system(s) that mediated the effect were to be determined. A pharmacologic approach was used. Male guinea pig gallbladder strips were placed in in vitro chambers filled with Krebs solution, maintained at 37°C, and gassed with 95% O2–5% CO2. Changes in tension were recorded using a polygraph.It was shown that the PKA/cAMP second messenger system mediated part of the observed chrysin-induced relaxation of cholecystokinin-induced tension, the PKC system also mediated part of the relaxation, and the inhibition of both extracellular Ca2+ entry and intracellular Ca2+ release also mediated the chrysin-induced relaxation. This is the first report of chrysin having an effect on gallbladder smooth muscle contraction.

A comparison of the effects of various sex steroids on cholecystokinin- and KCl-induced tension in female guinea pig gallbladder strips

Estrogen (E) has been shown to have an inhibitory effect on the contractility of gastrointestinal smooth muscle, including the gallbladder. During pregnancy E and progesterone (P) levels are elevated. A biliary stasis may develop during pregnancy that is characterized by an increase in the fasting and residual volumes and by a decrease in emptying capacity. This study investigates the effect of 17β-estradiol (E2), dihydrotestosterone (DHT), P, 17-hydroxyprogesterone (17-P), and a P metabolite, 20α-hydroxyprogesterone (20-P) on contraction in female guinea pig gallbladder strips. DHT, P, 17-P, 20-P, and E2 each induced a concentration-dependent relaxation of cholecystokinin octapeptide (CCK) induced tension. DHT, E2, and P also induced a concentration-dependent relaxation of KCl-induced tension. When the response to E2 was compared to strips from young female guinea pigs with those taken from guinea pigs in late pregnancy, there was no significant difference in the response to either 50 or 100μM E2; however, 10μM E2 caused a significant increase (p<0.05) in the amount of relaxation in strips from pregnant guinea pigs. Treatment of the strips from young guinea pigs with PKA inhibitor 14–22 amide myristolated had no significant effect on the E2-induced relaxation. Treatment of the strips with 2-APB, an inhibitor of IP3 induced Ca2+ release, produced a significant (p<0.001) increase in the amount of E2-induced relaxation when either CCK or KCl were used. Neither KT5823, a PKG inhibitor, nor L-NMMA, a nitric oxide (NO) synthase inhibitor, had a significant effect on the E2-induced relaxation. Bisindolymaleimide IV and chelerythrine Cl−, PKC blockers, were used in combination with no significant effect on the amount of CCK-induced tension, but significantly (p<0.01) increased the amount of E2-induced relaxation. When either E2 or P were added to the chambers 3min prior to either CCK or KCl, a significant decrease (p<0.001) in the amount of tension generated was observed. The inhibition of extracellular Ca2+ entry mediates both P-induced and E2-induced relaxation of CCK- and KCl-induced tension in female guinea pig gallbladder strips.

Chrysin relaxes cholecystokinin or KCl‐induced tension in male guinea pig gallbladder strips through multiple signaling pathways

The flavonoids are a large group of plant-derived phenolic compounds with biological effects including modulating vascular tone. The flavone, chrysin, has relaxant effects on vascular smooth muscle and intestinal smooth muscle. In this laboratory, chrysin relaxed cholecystokinin octapeptide (CCK) or KCl-induced tension. The relaxation was concentration dependent. An in vitro technique was used to determine which second messenger system(s) mediate the observed relaxation. Paired t-tests were used for statistical analysis. Differences between mean values with p<0.05 were considered significant. To determine if the PKA/cAMP second messenger system mediated the chrysin-induced relaxation, PKA inhibitor 14–22 amide myristolated (PKA-IM; 180 nM) was used. PKA-IM caused a significant (p<0.001) decrease in chrysin-induced relaxation (69.8±3.5 vs. 56.4±2.8%). When the PKC inhibitors bisindolymaleimide IV (0.5 μM) and chelerythrine Cl− (5 mM) were used together, a significant (p<0.01) reduction in chrysin-induced relaxation (57.9±4.4 vs. 52.2±5.1%) was observed. 2-APB (125 μM), an inhibitor of IP3 induced intracellular Ca2+, significantly (p<0.001) decreased the amount of chrysin-induced relaxation (60.7±6.0 vs. 49.1±2.5%). Genistein (10 μM), a protein tyrosine kinase inhibitor had no significant effect on the chrysin-induced relaxation. L-NMMA (20μM), a NO synthase inhibitor, had no significant effect on the amount of chrysin-induced relaxation. The results show that the chrysin-induced relaxation is mediated by the PKA/cAMP and PKC second messenger systems. The release of intracellular Ca2+ is also involved in mediating the chrysin effect. Since chrysin also relaxed the KCl-induced tension, extracellular Ca2+ entry is also involved in mediating the chrysin-induced relaxation. In conclusion, the chrysin-induced relaxation is mediated by multiple signaling pathways.

17β-Estradiol relaxes cholecystokinin- and KCl-induced tension in male guinea pig gallbladder strips

Estrogen has been shown to have an inhibitory effect on the contractility of gastrointestinal smooth muscle, including the gallbladder. Since estrogen and progesterone levels are elevated during pregnancy, a biliary stasis may develop during pregnancy that is characterized by an increase in the fasting and residual volumes and by a decrease in emptying capacity. This study investigates the effect of 17β-estradiol (E2) on contraction in male guinea pig gallbladder strips. E2 induced a concentration-dependent relaxation of either CCK-induced tension or KCl-induced tension. Pretreatment of the strips with PKA inhibitor 14–22 amide myristolated had no significant effect on the E2-induced relaxation. Pretreatment of strips with 2-APB, and inhibitor of IP 3 induced Ca 2+ release, produced a significant ( p < 0.001) increase in the amount of E2-induced relaxation when either CCK or KCl were used to induce tension. KT5823, an inhibitor of PKG, also significantly ( p < 0.001) increased the amount of E2-induced relaxation. Genistein, an inhibitor of protein tyrosine kinase, had no significant effect on the E2-induced relaxation. Bisindolymaleimide IV and chelerythrine Cl- when used in combination had no significant effect on the amount of CCK-induced tension, but significantly ( p < 0.001) increased the amount of E2-induced relaxation. When E2 was added to the chambers prior to either CCK or KCl, a significant decrease ( p < 0.001) in the amount of tension generated was observed. The inhibition of extracellular Ca 2+ entry mediates the E2-induced relaxation of CCK- and KCl-induced tension in male guinea pig gallbladder strips.

Evidence that actomyosin cross bridges contribute to “passive” tension in detrusor smooth muscle

Contraction of detrusor smooth muscle (DSM) at short muscle lengths generates a stiffness component we termed adjustable passive stiffness (APS) that is retained in tissues incubated in a Ca(2+)-free solution, shifts the DSM length-passive tension curve up and to the left, and is softened by muscle strain and release (strain softened). In the present study, we tested the hypothesis that APS is due to slowly cycling actomyosin cross bridges. APS and active tension produced by the stimulus, KCl, displayed similar length dependencies with identical optimum length values. The myosin II inhibitor blebbistatin relaxed active tension maintained during a KCl-induced contraction and the passive tension maintained during stress-relaxation induced by muscle stretch in a Ca(2+)-free solution. Passive tension was attributed to tension maintaining rather than tension developing cross bridges because tension did not recover after a rapid 10% stretch and release as it did during a KCl-induced contraction. APS generated by a KCl-induced contraction in intact tissues was preserved in tissues permeabilized with Triton X-100. Blebbistatin and the actin polymerization inhibitor latrunculin-B reduced the degree of APS generated by a KCl-induced contraction. The degree of APS generated by KCl was inhibited to a greater degree than was the peak KCl-induced tension by rhoA kinase and cyclooxygenase inhibitors. These data support the hypothesis that APS is due to slowly cycling actomyosin cross bridges and suggest that cross bridges may play a novel role in DSM that uniquely serves to ensure proper contractile function over an extreme working length range.

Effects of carbon monoxide on trout and lamprey vessels

Carbon monoxide (CO) is endogenously produced by heme oxygenase (HO) and is involved in vascular, neural, and inflammatory responses in mammals. However, the biological activities of CO in nonmammalian vertebrates is unknown. To this extent, we used smooth muscle myography to investigate the effects of exogenously applied CO (delivered via a water-soluble CO-releasing molecule, CORM-3) on isolated lamprey (Petromyzon marinus) dorsal aortas and examined its mechanisms of action on trout (Oncorhynchus mykiss) efferent branchial (EBA) and celiacomesenteric (CMA) arteries. CORM-3 dose-dependently relaxed all vessels examined. Trout EBA were twofold more sensitive to CORM-3 when precontracted with norepinephrine (NE) than KCl and CORM-3 relaxed five-fold more of the NE- than KCl-induced tension. Glybenclamide (10 microM), an ATP-sensitive potassium channel inhibitor, inhibited NE-induced contraction, but did not affect CORM-3-induced relaxation. NS-2028 (10 microM), a soluble guanylyl cyclase inhibitor, had no effect on a NE-contraction, but inhibited a subsequent CORM-3-induced relaxation. Zinc protopophyrin-IX (ZnPP-IX, 0.3-30 microM), a HO inhibitor, elicited a small, yet dose-dependent and significant, increase in baseline tension but did not have any effect on subsequent NE-induced contractions or a nitric oxide-induced relaxation (via sodium nitroprusside). [ZnPP-IX] greater than 3 microM, however, significantly reduced the predominant vasodilatory response of trout EBA to hydrogen sulfide. These results implicate an active HO/CO pathway in trout vessels having an impact on resting vessel tone and CO-induced vasoactivity that is at least partially mediated by soluble guanylyl cyclase.

Effects of carbon monoxide on sea lamprey and rainbow trout systemic arteries

Carbon monoxide (CO) is an endogenously produced gas involved in vascular, neural, and inflammatory responses in mammals. CO activity in non‐mammalian vertebrates, however, is unknown. To this extent, we utilized smooth muscle myography to investigate the effects of exogenously applied CO (via CORM‐3) on isolated lamprey (Petromyzon marinus) dorsal aortas and trout (Oncorhynchus mykiss) efferent branchial (EBA) and celiacomesenteric (CMA) arteries. CORM dose‐dependently relaxed norepinephrine‐ (NE) and U46619‐, thromboxane‐A2 mimetic, ‐induced contractions of lamprey dorsal aortas. CORM relaxed both NE (10μM)‐and KCl (80mM)‐ prestimulated trout EBA and CMA dose dependently. EBA were 2‐fold more sensitive to CORM when prestimulated with NE than KCl and CORM relaxed 5‐fold more of the NE‐ than KCl‐induced tension. Glybenclamide (lOμM) inhibited a NE‐induced contraction, but did not effect a CORM induced relaxation. NS‐2028 (10μM), a solubule guanylyl cyclase inhibitor, had no effect on a NE‐contraction, but inhibited a subsequent CORM‐induced relaxation. Zinc protopophyrin‐IX (30μM), a heme‐oxygenase (HO) inhibitor, elicited a significant increase in baseline tension and eliminated the tri‐phasic EBA response to Na2S. These results implicate an active HO/CO pathway in trout vessels on resting vessel tonus and H2S‐induced vasoactivity that is partially mediated by soluble guanylyl cyclase.

Chronic withdrawal of a high-palatable obesity-inducing diet completely reverses metabolic and vascular abnormalities associated with dietary-obesity in the rat

Chronic consumption of a high-palatable diet induces obesity and markedly impairs arterial relaxation. We have recently reported that endothelial function is only partially resorted after acute withdrawal of palatable diet. Therefore, this study was designed to investigate the effects of chronic withdrawal of high-palatable obesity-inducing diet on metabolic and vascular function in rats. Wistar rats were fed either standard laboratory chow throughout (controls) or given a highly-palatable diet (diet-fed) for 15 weeks; or fed the diet for 8 weeks and then returned to chow (diet-to-chow) for further 7 weeks before sacrifice. Diet-fed rats had higher body weight, fat mass, liver and heart weight than both chow-fed and diet-to-chow groups ( P<0.01 for all). Compared with chow-fed and diet-to-chow groups, diet-fed rats had significantly raised fasting plasma levels of insulin, leptin and triglycerides levels (each +180%; P<0.0001), but not glucose or non-esterified fatty acids. There were no significant differences between any metabolic parameters between chow-fed and diet-to-chow groups. Mesenteric arteries showed no significant differences between any groups in KCl-induced tension generation, while diet-fed groups had significantly higher noradrenaline-induced vasoconstriction than both chow-fed and diet-to-chow groups. Maximum endothelial-dependent vasorelaxation responses to carbamylcholine (CCh) were significantly (by 23%; P<0.001) attenuated in the diet-fed group. This defect was abolished in the diet-to-chow group. There were no significant differences in endothelium-independent vasorelaxation responses to sodium nitroprusside between the three groups. In conclusion, palatable diet induces obesity and metabolic abnormalities as well as a marked endothelial dysfunction. These abnormalities are completely reversed by chronic withdrawal of the obesity-inducing high-palatable diet.

ALIPHATIC CHLORINATED HYDROCARBONS ALTER THE CONTRACTILE RESPONSES OF TRACHEAL SMOOTH MUSCLE IN PIGLET

Aliphatic chlorinated hydrocarbons (ACHs) are widely used in several industrial processes and are also found in many commercial household products. They are classified as hazardous air pollutants, since ACHs exposure induces respiratory complications including airway hyperactivity. However, the contribution of airway smooth muscle tone to ACH-induced respiratory dysfunction has not been elucidated. Thus, the effects of ACHs such as dichloromethane (DCM), dichloroethane (DCE), and trichloromethane (TCM), on the basal and stimulant-induced contractile responses in piglet tracheal smooth muscle were investigated. ACHs at 100-1000 ppm were found to evoke the basal contraction of tracheal smooth muscle strips. Although DCM, DCE, and TCM enhanced the muscle tone precontracted by KCl, they exerted differential effects on acetylcholine- or histamineinduced muscle contraction. DCE did not alter the muscle tone activated by acetylcholine and histamine. DCM at 1000 ppm enhanced the muscle tension precontracted by acetylcholine but not by histamine. TCM at 30-1000 ppm increased the histamine-induced muscle contraction, but at 1000 ppm relaxed the muscle precontracted by acetylcholine. DCE and TCM at the highest concentration (1000 ppm) provoked a biphasic response with an initial increase in KCl-induced muscle tension followed by a decrease. Furthermore, pretreatment with DCE potentiated the acetylcholine-, histamine-, and KCl-induced muscle contractile responses. Pretreatment with TCM potentiated the histamine-, and KCl-induced response, but DCM only potentiated the KCl-induced response. The results suggest that ACH exposure altering the basal and spasmogen-induced contractile responses might participate in airway impairment with hyperresponsiveness.

Relaxation of human ureteral smooth muscle in vitro by modulation of cyclic nucleotide-dependent pathways.

Phosphodiesterases (PDE) are key enzymes regulating intracellular cyclic nucleotide turnover and, thus, smooth muscle tension. Recent reports have indicated the presence of PDE isoenzymes 1, 2, 4, and 5 in cytosolic supernatants prepared from human ureteral smooth muscle homogenates and the ability of second-generation inhibitors of PDE 3, 4, and 5 to relax KCl-induced tension of human ureteral muscle in vitro. The aim of the present study was to evaluate the functional effects of recently developed, third-generation isoenzyme-selective PDE inhibitors, the nitric oxide (NO)-donating agents sodium nitroprusside (SNP) and dihydropyridine (DHP), which is also described as an antagonist of L-type calcium channels, and the adenylyl cyclase-stimulating drug forskolin on tissue tension and cyclic nucleotide levels of human ureteral smooth muscle segments in vitro. Relaxant responses of human ureteral smooth muscle were investigated in vitro using the organ bath technique. Cyclic nucleotides cAMP and cGMP were determined by specific radioimmunoassay following time and dose-dependent incubation of the ureteral tissue with the drugs. The most pronounced relaxing effects on KCl-induced tension of ureteral smooth muscle were exerted by nitrovasodilator SNP, PDE4 inhibitor rolipram, and PDE5 inhibitors E 4021 and morpholinosulfonyl-pyrazolopyrimidine (MSPP). Relaxing potency of the drugs was paralleled by their ability to elevate intracellular levels of cGMP and cAMP, respectively. Our data suggest the possibility of using selective inhibitors of PDE isoenzymes 4 and 5 in the treatment of ureteral stones and ureteral colic.

EFFECT OF MAGNESIUM SULFATE ON LEUKOTRIENE-INDUCED AIRWAY TONE. 1789

Magnesium sulfate (MgSO4) may be useful in patients failing conventional therapy for asthma. Studies suggest that Mg2+ is a voltage-dependent Ca2+ channel (VDC) antagonist, causing greater relaxation in airways contracted with KCl than with cholinergic agonists. This is consistent with the fact that in KCl-induced tension, both initiation and maintenance of contraction require Ca2+ entry through VDCs, but in cholinergic-induced tension, initiation of contraction is mediated by receptor-operated Ca2+ channels, and only maintenance requires VDCs. However, there is little evidence showing involvement of VDCs in airway contraction to physiologic agonists, such as leukotriene D4 (LTD4), an inflammatory mediator in asthma. The mechanism of LTD4-mediated airway contraction is not precisely known. Thus, this study was designed to evaluate the effect of MgSO4 on LTD4-induced airway tone. Methods. Newborn guinea pig tracheal ring segments (n=7-9) were constricted to 2 grams tension with acetylcholine (ACh), KCl, or LTD4. Changes in isometric tension to cumulative additions of MgSO4 (1.2-11.2 mM) were measured. Results. After constriction with ACh, MgSO4 produced dose-dependent airway relaxation, at maximum by 33±6%. After constriction with LTD4, MgSO4 relaxed airway tone by 42±8%, similar to that produced after constriction with ACh (p=NS). However, after constriction with KCl, MgSO4 produced greater relaxation (74±7%, p<0.0001). Conclusions. MgSO4 significantly relaxes leukotriene-induced airway contraction in vitro, supporting its use clinically in reducing inflammatory-mediated airway hyperresponsiveness. The similarity of relaxation responses after constriction with ACh and LTD4 suggests that, as with ACh, maintenance of contraction to LTD4 may require VDCs.

Nitric oxide has no chronotropic effect in right atria isolated from rat heart

This study was designed to determine if nitric oxide (NO) has direct effects on heart rate or if it is involved in the chronotropic actions of adrenergic or cholinergic stimulation. Right atria were isolated from hearts of adult male rats, bathed in Krebs-Henseleit buffer (37°C), and used to monitor spontaneous rate. For comparison, ring segments of thoracic aorta were also suspended in the Krebs-Henseleit solution and used to examine vascular actions of various agents. The dose-dependent chronotropic effects of acetylcholine (10 −7–10 −3 M) and norepinephrine (10 −8−3 × 10 −4 M) in right atria were not affected by pretreatment with 10 −4 M N- nitro- L-arginine or 10 −3 M N- nitro- L-arginine-methyl ester, inhibitors of l-arginine-derived NO production. SIN-1 (3-morpholino-sydnonimine), an agent which releases NO in aqueous solution, elicited a dose-dependent (0.3–100 μM) vasorelaxation in aortic preparations constricted with 60 mM KCl; the ED 50 value for this effect was increased by pretreatment with methylene blue (10 μM) and LY-83,583 (6-(phenylamino)-5,8-quinolinedione; 1 and 3 μM), compounds which inhibit NO-induced stimulation of guanylate cyclase. SIN-1 produced a negative chronotropic effect in right atria; however, this action was not observed at concentrations less than 300 μM and was not antagonized by methylene blue or LY-83,583. 8-Bromo cyclic GMP produced a dose-dependent (10–3000 μM) decrease in KCl-induced tension in aortic rings. In right atria, 8-bromo cyclic GMP elicited a positive chronotropic effect. These results suggest that NO has no chronotropic action in rat atrial preparations.

Aging differentially modifies arterial sensitivity to endothelin-1 and 5-hydroxytryptamine: Studies in dog coronary arteries and rat arterial mesenteric bed

The influence of age on vascular reactivity to endothelin-1 (ET-1) and 5-hydroxytryptamine (5-HT) was studied in coronary artery rings from dogs of 9 years of age or younger, and dogs older than 9 years. ET-1 caused concentration-dependent contractions that developed about 100% of the 70 m M KCl-induced tension in the younger dogs; those from older dogs did not generate more than 20%. In contrast, 5-HT developed only about 20% of the KCl-induced tension in rings from young dogs, whereas in the older animals, it developed up to 120% of the KCl tension. No significant difference in the tension developed by 70 m M KCl was noted between both groups of dogs. Mechanical denudation of the endothelial cell layer caused a modest, yet significant, leftward shift of the ET-1 and 5-HT concentration-response curves only in the younger dogs. Nω- Nitro- l-arginine (15 μM) shifted the ET-1 concentration-response curves to the left in rings from both groups of dogs. Rings precontracted with 20 m M KCl relaxed in a concentration-dependent fashion with acetylcholine; its sensitivity was about threefold less in the older group of dogs. To validate the changes in vascular reactivity with age, a parallel study was performed perfusing the arterial mesenteric bed of rats of 3, 7, and 30 weeks of age. In this experimental model, the efficacy of ET-1 significantly decreased with age and that of 5-HT was significantly increased. The vasomotor reactivity of noradrenaline was modestly affected by aging, whereas the acetylcholine-induced vasorelaxation was significantly reduced with age.

Intracellular Mechanisms of Halothane's Effect on Isolated Aortic Strips of the Rabbit

The intracellular mechanisms of halothane action were examined in vascular smooth muscle from the aorta. Medial layers of the aorta from rabbits were mounted on photodiode tension transducers, stretched to 20 mg resting tension, and "skinned" with saponin. The skinned fiber preparations were then immersed in bathing solutions to study the effects of halothane (0.5-2%) on Ca2+ activation of the contractile proteins, and Ca2+ uptake and release from the sarcoplasmic reticulum (SR) using caffeine-induced tension transients. For comparison, isolated intact aortic rings were mounted on Blinks' dual tissue bath and attached to force transducers. The preparations were contracted with either 40 mM KCl, or norepinephrine (NE) followed by acetylcholine (ACh)- or sodium nitroprusside (SNP)-induced relaxation. At steady state contraction or relaxation, the effects of halothane (1-3%) were studied. The steady state tension during halothane was expressed as a percentage of the steady state tension before administration of halothane. In the isolated intact aortic rings, halothane (1-3%) produced biphasic effects on KCl-induced tension, i.e., an initially slight increase followed by decreases, independent of endothelium. Halothane markedly increased tension in the ACh- or SNP-relaxed state. The effects were dose-dependent. In the skinned aortic strips, halothane slightly decreased maximum Ca2+-activated tension development of the contractile proteins. Halothane decreased Ca2+ accumulation in the SR and increased Ca2+ release from the SR in a dose-dependent manner. The halothane-induced increases in Ca2+ release from the SR were blocked by ryanodine, an SR Ca2+ release channel blocker. It is concluded that halothane directly causes vascular contraction or relaxation, depending on the condition, and that halothane's effects on the SR may play a role.