Jack Diamond

Evidence that spontaneous contractile activity in the rat myometrium is not inhibited by NO-mediated increases in tissue levels of cyclic GMP.

1 There is conflicting evidence in the literature concerning the role of cyclic GMP in the regulation of myometrial contractility and the importance of hormonal status on the uterine response to cyclic GMP‐elevating agents. The objective of the present study was to investigate further the importance of cyclic GMP in the control of uterine contractility, by monitoring the effects of cyclic GMP‐elevating agents on spontaneous contractions and cyclic GMP levels in myometrial strips from pregnant rats and from ovariectomized rats under the influence of oestrogen and/or progesterone. 2 Sodium nitroprusside (SNP) 5 mM, atrial natriuretic peptide (ANP) 100 nM, L‐arginine 1 mM and 8‐bromo‐cyclic GMP 100 mM had no relaxant effect on the spontaneous contractions of myometria from pregnant rats or from ovariectomized rats under the influence of oestrogen or progesterone. 3 Tissue levels of cyclic GMP were significantly elevated by SNP in all treatment groups, including pregnant animals. For example, in ovariectomized, progesterone‐treated rats, SNP raised cyclic GMP levels approximately 8 fold from a basal level of 2.9±0.4 pmol mg−1 protein to 24.8±4.0 pmol mg−1 protein. ANP increased cyclic GMP levels approximately 2 fold in all treatment groups, except in the pregnant animals. L‐Arginine elevated cyclic GMP significantly only in ovariectomized, vehicle‐treated myometria. 4 The activity of cyclic GMP‐dependent protein kinase (PKG) was significantly increased (3 fold) in myometria exposed to SNP (5 mM). Thus, the inability of SNP to relax uterine preparations was not due to a failure of SNP‐elevated cyclic GMP to activate PKG. 5 The more potent NO donor, S‐nitroso‐N‐acetylpenicillamine (SNAP), at a concentration of 100 μM was able to inhibit spontaneous contractions significantly in myometrial preparations from both non‐ovariectomized and ovariectomized rats treated with oestrogen or progesterone. 6 Tissue levels of cyclic GMP were markedly increased by SNAP at concentrations of 10, 30 and 100 μM. At 100 μM, cyclic GMP levels increased from 1.9±0.2 pmol mg−1 protein to 74.0±18.0 pmol mg−1 protein. However, complete or partial blockade of SNAP‐induced increases in cyclic GMP levels by the soluble guanylyl cyclase inhibitor, ODQ (25 μM), had no effect on the relaxant response to SNAP. Thus, the relaxant effect of SNAP in this tissue appears to be mediated via a mechanism independent of cyclic GMP. 7 Taken as a whole, the results of the present study indicate that cyclic GMP does not play an important role in the control of contractility in the rat uterus.

Evaluation of Kemptide, a synthetic serinecontaining heptapeptide, as a phosphate acceptor for the estimation of cyclic AMP-dependent protein kinase activity in respiratory tissues

In the search for a phosphate acceptor to estimate cyclic AMP-dependent protein kinase (A-kinase) activity in respiratory tissues devoid of the disadvantages inherent with the use of histones, we compared and contrasted a conventional substrate, histone IIa, with that of a novel heptapeptide phosphate acceptor, Kemptide (Kemp et al., J Biol Chem 252: 4888-4894, 1977). The specific activities of soluble A-kinase isolated from guinea-pig lung parenchyma and from bovine tracheal smooth muscle were significantly (12-16-fold) greater when Kemptide was used as substrate when compared to histone IIa. Moreover, studies with a specific inhibitor of A-kinase demonstrated that whilst the cyclic AMP-stimulated phosphorylation of Kemptide was catalysed exclusively by A-kinase, only approximately 89% of the phosphate incorporated into histone IIa was attributed to the activity of this enzyme. The activation constant (Kact) of cyclic AMP for A-kinase was estimated to be 1.5 microM when Kemptide was used as substrate, 25-fold higher than that obtained using histone IIa (60 nM) under identical conditions. In a complementary series of experiments it was found that both the basal and isoprenaline-stimulated A-kinase activity ratios were significantly higher when histone IIa was used as substrate when compared with that obtained using Kemptide. Elevating the ionic strength of the homogenization buffer or assay cocktail with NaCl produced a reversible (following dialysis), concentration-dependent, mixed uncompetitive, inhibition of cyclic AMP-stimulated histone phosphorylation; phosphokemptide formation was unaffected. The ability of salt to inhibit A-kinase activity also affected the elution profile of A-kinase isoenzymes following their separation by DEAE-cellulose chromatography. With histone IIa as substrate the Type I:Type II isoenzyme ratio in guinea-pig lung and in bovine tracheal smooth muscle was 9:91 and 35:65, respectively. Changing the phosphate acceptor to Kemptide reduced these respective ratios to 5:95 and 29:71. Although the effect on the isoenzyme ratio was small, the specific activities of the two isoenzymes isolated from both tissues were markedly attentuated by ca. 9% (Type I) and 36% (Type II). It is concluded that Kemptide is without many of the disadvantages inherent with histone IIa and is, thus, a preferable phosphate acceptor for estimating soluble A-kinase activity and determining the isoenzyme ratio in guinea-pig lung and in bovine tracheal smooth muscle.

The effect of M&B 22948 on methacholine- and histamine-induced contraction and inositol 1,4,5-trisphosphate levels in guinea-pig tracheal tissue.

The effect of a cyclic GMP phosphodiesterase inhibitor, M&B 22948, on methacholine‐ and histamine‐induced contraction and inositol 1,4,5‐trisphosphate (IP3) elevation was studied in guinea‐pig tracheal rings. After addition of methacholine or histamine the rise in IP3 content was rapid and transient reaching a maximum after 5–15 s, which coincided with the maximum rate of tension development. Cyclic GMP levels of the tissue were elevated by M&B 22948 before agonist stimulation and further elevated by addition of methacholine or histamine. Cyclic AMP levels were not altered by any of these agents. M&B 22948 abolished IP3 generation induced by methacholine or histamine, but did not alter the rate or magnitude of tension development. Thus, IP3 generation does not appear to be responsible for the contractions induced by methacholine or histamine in this tissue.

Elevation of cyclic AMP by prostacyclin is accompanied by relaxation of bovine coronary arteries and contraction of rabbit aortic rings

The role of cyclic AMP (cAMP) in the control of vascular smooth muscle tension was examined by comparing the effects of prostacyclin (PGI2) on tension and cAMP levels in helical strips of bovine coronary arteries and in rabbit aortic rings, both denuded of endothelium. In bovine coronary arteries, PGI2 elevated cAMP levels and relaxed the muscles. The PGI2-induced cAMP elevation preceded the relaxation and both parameters were altered in a dose-dependent manner by increasing concentrations of PGI2 (0.3, 3 and 30 microM). These results are consistent with a role for cAMP as a mediator of vascular smooth muscle relaxation. Cyclic AMP levels were also elevated by PGI2 in a concentration- and time-dependent manner in rabbit aortic rings. However, in direct contrast to the results in the bovine coronary arteries, PGI2-induced elevation of cAMP in the aortic rings was accompanied by contraction rather than relaxation. Isoproterenol, a drug which is generally believed to relax smooth muscles by virtue of its ability to increase tissue levels of cAMP, relaxed PGI2-contracted aortic rings with no further elevation of cAMP beyond that caused by the PGI2 alone. These results demonstrate that cAMP elevation and relaxation of vascular smooth muscle are not always well correlated. It is possible that some form of functional compartmentalization of cAMP or cAMP-dependent protein kinase exists in these tissues.

Translocation of protein kinase C in bovine tracheal smooth muscle strips: the effect of methacholine and isoprenaline.

Investigations into the mechanisms involved in the contraction of smooth muscle have suggested that the generation of diacylglycerol and the activation of protein kinase C (PKC) may be important in the generation or maintenance of smooth muscle tone. The present study examined the possible role of PKC in the contraction of bovine tracheal smooth muscle. Methacholine (10 microM) induced a rapid elevation in PKC activity associated with the membrane fraction. PKC levels were significantly elevated in the membrane fraction 30 s after agonist addition, reached a maximum at 1 min and then declined to and remained at a lower level which was still elevated above basal. A concomitant decrease in cytosolic PKC activity of smaller magnitude was observed during this period of stimulation. This methacholine-induced re-distribution of PKC from the cytosol to the membrane was concentration-dependent and was blocked by atropine. Pre-treatment of tissues for 2 min with 100 microM isoprenaline prevented both the re-distribution of PKC and the contraction produced by 1 microM methacholine. Addition of 1 microM isoprenaline to tissues pre-contracted with 1 microM methacholine reversed the re-distribution of PKC produced during contraction and completely relaxed the tissues. Thus, under these conditions, translocation of PKC from the cytosol to the membrane seems to be well correlated with contractions of bovine tracheal smooth muscle. Whether the PKC translocation is responsible for the observed changes in muscle tone or whether the enzyme translocation is a result of drug-induced changes in [Ca2+]i, remains to be determined.

Effects of prostaglandin E1, Isoproterenol and forskolin on cyclic AMP levels and tension in rabbit aortic rings

The role of cyclic AMP in the control of vascular smooth muscle tone was studied by monitoring the effects of prostaglandin E1 (PGE1), isoproterenol and forskolin on cyclic AMP levels and tension in rabbit aortic rings. PGE1, isoproterenol and forskolin all increased cyclic AMP levels in rabbit aortic rings. Isoproterenol and forskolin relaxed phenylephrine-contracted aortic rings, but PGE1 contracted the rings in the presence or absence of phenylephrine. Isoproterenol relaxed these PGE1-contracted aortic rings without further change in total cyclic AMP levels, which were already elevated by the PGE1 alone. Pretreatment with forskolin potentiated the effects of PGE1 on cyclic AMP levels. PGE1 caused contractions in muscles partially relaxed by forskolin, even though very large increases in cyclic AMP levels (30 fold) were produced by PGE1 in the presence of forskolin. Isoproterenol was able to relax these forskolin-treated, PGE1-contracted muscles with no further increase in cyclic AMP levels. Thus, there does not appear to be a good correlation between total tissue levels of cyclic AMP and tension in these experiments. Our results suggest that, if cyclic AMP is responsible for relaxation of smooth muscle, some form of functional compartmentalization of cyclic AMP must exist in this tissue.

Cyclic GMP‐dependent protein kinase activation in the absence of negative inotropic effects in the rat ventricle

1 It has been suggested that activation of cyclic GMP‐dependent protein kinase (PKG) is a necessary step in the chain of events leading to the production of negative inotropy by muscarinic receptor agonists in mammalian ventricles, and that some cyclic GMP‐elevating agents, such as sodium nitroprusside (SNP), fail to exert a negative inotropic effect because they elevate cyclic GMP levels in a pool that does not activate the kinase. This hypothesis was tested in the present study by monitoring the effects of carbachol, SNP and atrial natriuretic peptide (ANP) on contractility, cyclic GMP content and PKG activity in rat intact ventricular preparations and freshly isolated ventricular cardiomyocytes. 2 The presence of PKG in both the intact vehicle and in isolated ventricular cardiomyocytes was confirmed by MonoQ anion exchange chromatography and Western blotting. The elution profile indicated that the conditions of the PKG assay were selective for measuring PKG activity. 3 Carbachol induced a marked negative inotropic effect in intact, perfused hearts and ventricular strips in the presence of isoproterenol. The negative inotropic effect of carbachol was not associated with significant changes in cyclic GMP content or PKG activity in intact ventricular tissue, or in PKG activity in isolated cardiomyocytes. 4 SNP and ANP significantly increased cyclic GMP levels and activated PKG in intact ventricular preparations. Both drugs also activated PKG in isolated cardiomyocytes. However, neither drug had any negative inotropic effect in isoprenaline‐stimulated perfused hearts and ANP did not change the contractility of isoprenaline‐stimulated isolated cardiomyocytes. 5 The results of this study demonstrate that the negative inotropic effects of muscarinic receptor agonists can occur in the absence of significant activation of PKG. Conversely, marked increases in ventricular cyclic GMP content and PKG activity caused by SNP or ANP were not accompanied by a negative inotropic effect. 6 These results suggest that increases in cyclic GMP levels and activation of PKG do not play important roles in the regulation of rat ventricular contractility by muscarinic receptor agonists.

β-Adrenoceptors Cyclic AMP, and Cyclic GMP in Control of Uterine Motility

Adenosine 3′,5′-cyclic monophosphate (cAMP) and the enzymes necessary for its synthesis and degradation have been shown to be present in most mammalian cells, including myometrial cells. Cyclic AMP is considered to be the “second messenger” responsible for mediating the actions of numerous drugs and hormones, and such a role has been suggested for this cyclic nucleotide in the uterine relaxant effects of β-adrenoceptor agonists such as isoproterenol and epinephrine. Some of the evidence for and against a role for cAMP as a mediator of the uterine relaxant effects of β agonists is discussed in the following sections. By way of background, brief descriptions of the roles of adrenergic receptors in control of uterine motility and the effects of estrogen and progesterone on these responses are included. The factors involved in coupling adrenergic receptors to the cAMP system are also discussed.

Relationship between contraction and cyclic GMP levels of Guinea pig taenia coli

The relationship between isometric tension and the increase in cyclic GMP level associated with the initial phase of drug-induced contraction of taenia coli was studied. Maximal contraction induced by 100 micrometers carbachol or 124 mM KCl occurred in 30 sec and was associated with an increase in cyclic GMP levels of at least ten fold. However, 100 micrometers serotonin or low concentrations of carbachol (30 microM) produced nearly maximal contraction but only 10--20% of the maximal elevation in cyclic GMP induced by high concentrations of KCl. Furthermore, in the presence of a low concentration of Ca2+, or verapamil, both 124 mM KCl and 100 micrometers carbachol increased cyclic GMP with little elevation in isometric tension. Thus, large contractions were obtained with only small increases in cyclic GMP, and under other conditions, large increases in cyclic GMP were obtained with little increase in isometric tension. The results suggest that the initial increase in cyclic GMP level caused by these agents is not directly dependent on Ca2+ in the cellular pool containing the contractile proteins.

The effect of Ca2+ on the translocation of protein kinase C in bovine tracheal smooth muscle.

The role of calcium in protein kinase C redistribution was studied in bovine tracheal smooth muscle preparations contracted by methacholine. Previous results have shown that, in the presence of normal extracellular Ca2+, 10 microM methacholine produced a sustained contraction and a sustained translocation of protein kinase C from the cytosol to the membrane. In the present study, when tissues were preincubated in Ca(2+)-free buffer containing 1 mM EGTA, methacholine produced a rapid but transient elevation in membrane-associated protein kinase C activity which was detected at 30 s and had returned to basal within 20 min. The redistribution of protein kinase C from the cytosol to the membrane induced by 1 microM methacholine in normal Ca2+ was reversed by removal of the extracellular Ca2+ and addition of 2 mM EGTA during agonist stimulation. Removal of the Ca2+ caused approximately 50% relaxation after 10 min. Verapamil (30 microM) partially reversed the methacholine-induced protein kinase C redistribution and caused approximately 40% relaxation after 15 min. Sodium nitroprusside (10 microM) caused a rapid relaxation and complete reversal of the protein kinase C redistribution induced by methacholine. High K+ (60 mM) also induced a sustained contraction and redistribution of protein kinase C from the cytosol to the membrane. Suitable antagonists were added to the bathing medium to block the effects of endogenous mediators which could be released by KCl-induced depolarization. Thus, translocation of protein kinase C is obtained in the absence of receptor activation.(ABSTRACT TRUNCATED AT 250 WORDS)