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J N Wells

Publications and source records attributed to J N Wells.

At least 37 records · Page 2Linked to original sources
PubMed1989

Endogenous adenosine restrains renin release during sodium restriction.

The purpose of this study was to determine the role of endogenous adenosine in controlling renin release during both a normal and low sodium diet. To probe the involvement of endogenous adenosine in the control of renin release, we examined the effects of an adenosine receptor antagonist, 1,3-dipropyl-8-(p-sulfophenyl)xanthine (DPSPX), on renin release in rats fed either a normal or low sodium diet. All studies were conducted in the in situ autoperfused kidney. DPSPX significantly increased arterial and renal venous levels of renin in both groups of animals; however, statistical analysis of the data (2-factor analysis of variance) indicated that DPSPX increased aortic and renal venous levels of renin more in rats fed a low sodium diet compared to rats fed a normal sodium diet. Also, whereas DPSPX did not significantly increase the venoarterial difference of renin activity across the kidney or the calculated net secretion rate of renin in rats on a normal sodium diet, both of these indices of renin release were significantly increased by DPSPX in rats on a low sodium diet. The effects of DPSPX on renin release could not be explained by changes in renal hemodynamics or excretory function. Additional experiments with rats on a low sodium diet that were treated with propranolol demonstrated that the effects of DPSPX on renin release were independent of the sympathetic nervous system.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine↗
PubMed1988

A novel mechanism of soluble guanylate cyclase stimulation: time-dependent activation by bacterial lipopolysaccharide in rat fetal spleen cells.

Small amounts of bacterial lipopolysaccharide (LPS) greatly increase cGMP levels in short term cultures of rat fetal liver and spleen cells in a dose and time dependent manner. To determine the role of guanylate cyclase in this response, a series of experiments was undertaken using either intact or broken fetal spleen cells, the most sensitive tissue evaluated to date. The phosphodiesterase inhibitor, 1-methyl-3-isobutyl-xanthine, potentiated the LPS-cGMP effect in cultures of these cells even at maximal doses of LPS. Moreover, after incubation of intact cells with LPS for 4 h, soluble guanylate cyclase (EC 4.6.1.2) activity was increased 2-fold, whereas particulate activity was unchanged. This increase in soluble activity was proportional to the dose of LPS, was synchronous with the elevation of cGMP levels, and was not associated with any change in cGMP-phosphodiesterase (EC 3.1.4.17) activity. In contrast to intact cells, neither total nor soluble guanylate cyclase activity was increased by the addition of LPS to spleen cell whole sonicate or cytosol for various times from 10 min to 3.5 h. These results suggest that the LPS-cGMP response is due to a persistent indirect stimulation of soluble guanylate cyclase activity that is both dose and time dependent.

1-Methyl-3-isobutylxanthine↗
PubMed1988

Biochemical events associated with activation of smooth muscle contraction.

Biochemical events associated with activation of smooth muscle contraction were studied in neurally stimulated bovine tracheal smooth muscle. A latency period of 500 ms preceded increases in isometric force and myosin light chain phosphorylation. However, stimulation resulted in the rapid hydrolysis of inositol phospholipids as demonstrated by increases in inositol phosphates by 500 ms. Inositol trisphosphate increased 2-fold with no significant change in inositol tetrakisphosphate. The apparent activation state of myosin light chain kinase was assessed indirectly through measurements of the fractional activation of a second calmodulin-dependent enzyme, cyclic nucleotide phosphodiesterase. The fractional activation of cyclic nucleotide phosphodiesterase increased after neural stimulation to a maximal extent by 500 ms and remained at this level for at least 4 s. The monophosphorylation of myosin light chain increased after 500 ms and reached a maximum value by 2 s. Diphosphorylation also occurred but to a much lesser extent. Fractional activation of cyclic nucleotide phosphodiesterase and myosin light chain phosphorylation both decreased after 10 min continuous stimulation, although the force response remained at a maximal level. These observations demonstrate that inositol trisphosphate formation and activation of cyclic nucleotide phosphodiesterase (and hence most likely myosin light chain kinase) by calmodulin precede myosin light chain phosphorylation and that these events are sufficiently rapid to mediate the contractile response of neurally stimulated tracheal smooth muscle.

Animals↗
PubMed1988

Involvement of a plasma membrane phosphodiesterase in the negative control of cyclic AMP levels by vasopressin in rat hepatocytes.

Vasopressin has been shown previously to lower the glucagon-induced increase of cyclic AMP levels in isolated rat hepatocytes by way of an enhanced phosphodiesterase (EC 3.1.4.17) activity. Five phosphodiesterase inhibitors were tested for their ability to prevent vasopressin from lowering cyclic AMP levels in intact hepatocytes and for their inhibitory effect in vitro on soluble and particulate phosphodiesterase activities partially purified from hepatocytes. Three soluble activities have been separated by DEAE-cellulose chromatography: a phosphodiesterase hydrolyzing both cyclic AMP and cyclic GMP, a form stimulated by cyclic GMP and a cyclic AMP-specific activity. The absence of any statistically significant correlation between the in vivo (in intact cells) and the in vitro (on isolated phosphodiesterases) potencies of the inhibitors does not support a role for the cytosolic phosphodiesterases in mediating the vasopressin-induced decrease in cyclic AMP levels. No statistically significant correlation was observed between the inhibition of the vasopressin effect on cyclic AMP accumulation and the inhibition of phosphodiesterase activity either associated with the native plasma membranes or solubilized from these membranes with 0.4 M NaCl. In contrast, a statistically significant correlation was observed between the degree of inhibition of the vasopressin effect in the intact cells and the degree of inhibition of the intrinsic phosphodiesterase still associated with the plasma membranes after high-salt treatment. These data indicate that a phosphodiesterase activity integral to the plasma membrane is very likely involved in the negative control of cyclic AMP levels by vasopressin.

Animals↗
PubMed1988

Relaxation of vascular and tracheal smooth muscle by cyclic nucleotide analogs that preferentially activate purified cGMP-dependent protein kinase.

Cyclic nucleotide analogs were used to study relaxation of pig coronary arteries and guinea pig tracheal smooth muscle in an attempt to determine the roles of cAMP- and cGMP-dependent protein kinases (cA-K and cG-K). In pig coronary artery strips, cGMP analogs were generally more effective than cAMP analogs in promoting relaxation of K+-induced contractions. Significant relaxation of this tissue was caused primarily by those cyclic nucleotide analogs that had high affinities for purified cG-K but not for cA-K. The low potencies of cA-K-specific analogs, as compared with cG-K-specific analogs, could not be readily explained by either unusually high susceptibilities to phosphodiesterases or low partition coefficients. The most potent cGMP analog, 8-(4-chlorophenylthio)-cGMP, exhibited a very slow reversibility of its relaxant effects in the intact tissue, consistent with its strong resistance to hydrolysis by phosphodiesterases measured in vitro. Pig coronaries contained atypically high levels of cGMP and cG-K, implying a potentially important role of this enzyme in smooth muscle function. Carbamylcholine-induced contractions of guinea pig tracheal segments were more sensitive than K+-induced pig coronary artery contractions to relaxation by cyclic nucleotide analogs. Consequently, the number of analogs that could be studied was significantly expanded. The cGMP analogs were again generally more potent, and the effectiveness of both cGMP and cAMP analogs in relaxing this preparation correlated with the Ka of the analogs for in vitro activation of cG-K, but not cA-K. A particularly strong correlation was observed when the effects of analogs modified only at the C-8 position were examined. A known target enzyme of cA-K, phosphorylase, was not activated by cG-K-specific analogs but was activated by high concentrations of the cA-K-specific analogs. Studies using cyclic nucleotide analogs support a role for cG-K, but not for cA-K, in decreasing smooth muscle tone.

Animals↗
PubMed1987

Effects of isoproterenol on active force and Ca2+ X calmodulin-sensitive phosphodiesterase activity in porcine coronary artery.

Relaxation of vascular smooth muscle following beta-adrenergic stimulation may result from reduction of the cytoplasmic Ca2+ concentration, reduction in the sensitivity of the contractile apparatus to Ca2+, or both. To help resolve these possibilities, we measured the extent of activation of Ca2+ X calmodulin-sensitive phosphodiesterase in intact porcine coronary artery strips as a functional indicator of the cytoplasmic Ca2+ concentration. Both calmodulin-stimulated phosphodiesterase activity and active force increased during K+ stimulation of coronary artery strips. Relaxation of K+-contracted artery strips following stimulus withdrawal was accompanied by rapid inactivation of Ca2+ X calmodulin-sensitive phosphodiesterase. The temporal relationship between isoproterenol-induced relaxation and inactivation of Ca2+ X calmodulin-sensitive phosphodiesterase was studied in both histamine- and K+-contracted tissues. Stimulation of strips with 10 microM histamine or with 44 mM K+ led to comparable increases both in active force and in calmodulin-stimulated phosphodiesterase activity. Thereafter, sustained contraction elicited by histamine was accompanied by a decrease in calmodulin-stimulated phosphodiesterase activity. Isoproterenol rapidly relaxed histamine-contracted strips and accelerated the decrease in phosphodiesterase activity. In contrast, sustained contraction in response to K+ was accompanied by a sustained elevation of calmodulin-stimulated phosphodiesterase activity. Isoproterenol treatment of K+-contracted tissues led to relaxation that was slow and incomplete, and it had very little effect on calmodulin-stimulated phosphodiesterase activity. We conclude that reduction of the cytoplasmic Ca2+ concentration is important for rapid relaxation of the coronary artery following beta-adrenergic stimulation. We cannot disallow the possibility that a decrease in the sensitivity of the contractile apparatus to Ca2+ is involved to some degree.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1987

Role of endogenous adenosine as a modulator of the renin response to salt restriction.

Numerous studies indicate that exogenous adenosine can inhibit renin release. However, the hypothesis that endogenous adenosine functions to restrain the renin response to physiological and/or pharmacological stimuli remains untested. To address this hypothesis, we examined the effects of a novel adenosine receptor antagonist, 1,3-dipropyl-8-para-sulfophenylxanthine (DPSPX), on renin release in rats on a normal versus a low salt diet. DPSPX did not affect renal blood flow, glomerular filtration rate, filtration fraction, urine volume, or sodium excretion in rats on either a normal or low salt diet. In contrast, in rats on a low salt diet, DPSPX significantly increased arterial and renal venous plasma renin activity and the gradient of plasma renin activity across the kidney. DPSPX did not alter these indices of renin release in rats on a normal salt diet. These data support the hypothesis that endogenous adenosine functions to restrain the renin response to salt depletion. Finally, if these findings are applicable to man, caffeine consumption could account for the variable antihypertensive effect of a low salt diet.

Adenosine↗
PubMed1987

Potent adenosine receptor antagonists that are selective for the A1 receptor subtype.

The xanthines currently represent the most potent class of adenosine receptor antagonists. However, known derivatives of xanthine show little difference in antagonist potency between the two putative adenosine receptor subtypes, A1 and A2. We conducted a systematic study of xanthine structure-activity relationships that compared antagonist potency at the A1 receptor of adipocytes with potency at the A2 receptor of platelets. Since adenosine receptors are coupled to adenylate cyclase in these tissues, inhibition of adenylate cyclase via A1 receptors and stimulation via A2 receptors were used as models of receptor activation. Antagonist potency was quantitated by Schild analysis, which yields an estimate of affinity (Ki) for the drug-receptor interaction. Ki values of a series of xanthine analogues enabled us to identify structural modifications than enhanced antagonist selectivity for one receptor subtype over the other. We found that changes in the substituent at position 8 of the xanthine nucleus influenced antagonist potency at the A1 adenosine receptor more than at the A2 receptor. In particular, an 8-cyclohexyl or 8-cyclopentyl substituent promoted antagonist selectivity for the A1 receptor subtype. Thus, 1,3-dipropyl-8-cyclopentylxanthine had comparatively high affinity (Ki = 0.47 +/- 2 nM) at the A1 receptor, and was roughly 150-fold more potent as an antagonist of the A1- than of the A2-adenosine receptor subtype. In addition, the cycloalkylxanthines were relatively ineffective as inhibitors of cyclic nucleotide phosphodiesterases when used at concentrations that produced marked adenosine receptor antagonism.

Adenosine↗
PubMed1986

Inhibition of cyclic nucleotide phosphodiesterases from pig coronary artery by benzo-separated analogues of 3-isobutyl-1-methylxanthine.

The linear and proximal benzo-separated analogues of 7-benzyl-3-isobutyl-1-methylxanthine, 3-isobutyl-1,8-dimethylxanthine, 3-isobutyl-8-tert-butyl-1-methylxanthine, 3-isobutyl-8-(methoxymethyl)-1-methylxanthine , and 1-isoamyl-3-isobutylxanthine have been prepared and assayed as inhibitors of the peak I and peak II forms of cyclic nucleotide phosphodiesterase from pig coronary artery. Most of the benzo analogues were less effective inhibitors of these phosphodiesterases when compared to 3-isobutyl-1-methylxanthine (IBMX) even though the active sites of both enzyme forms tolerated the stretched-out xanthines. Indeed, the linear benzo-separated analogue of 7-benzyl-IBMX was a more potent inhibitor of peak I activity than was IBMX.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Identification of the phosphodiesterase regulated by muscarinic cholinergic receptors of 1321N1 human astrocytoma cells.

Agonist occupation of muscarinic cholinergic receptors of 1321N1 human astrocytoma cells results in an activation of phosphodiesterase and a resultant 50-75% attenuation of isoproterenol-stimulated cyclic AMP accumulation. The effects of a series of phosphodiesterase inhibitors on muscarinic receptor-mediated inhibition of cyclic AMP accumulation and on the activities of partially purified, soluble phosphodiesterase have been compared to determine which form of phosphodiesterase activity is regulated by muscarinic receptors. The phosphodiesterase inhibitors (50 microM) 1-methyl-3-isobutylxanthine (MIX), 1-methyl-3-isobutyl-7-benzylxanthine (7-BzMIX), 1-methyl-3-isobutyl-8-methoxymethylxanthine (8-MeOMeMIX), and 2-O-propoxyphenyl-8-azapurin-6-one (MB 22948) blocked the effect of muscarinic receptor activation. However, 1-isoamyl-3-isobutylxanthine (IIX) and 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone (Ro 20-1724) did not block muscarinic receptor-mediated effects but enhanced isoproterenol-stimulated cyclic AMP accumulation. Three forms of soluble phosphodiesterase activity were resolved by DEAE-cellulose chromatography and sucrose density gradient centrifugation. A calmodulin-stimulated phosphodiesterase activity was inhibited by MIX, 7-BzMIX, 8-MeOMeMIX, and MB 22948 (IC50 values = 1-10 microM) but was not inhibited by IIX and Ro 20-1724. The similar relative capacities of the phosphodiesterase inhibitors for blocking both the muscarinic receptor-mediated attenuation of cyclic AMP accumulation and the calmodulin-stimulated phosphodiesterase activity in vitro suggest that it is this form of enzyme that is regulated by muscarinic receptor stimulation.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Irreversible inhibition of calmodulin-sensitive cyclic nucleotide phosphodiesterase.

Photolysis of solutions containing 4-azido-7-phenylpyrazolo-[1,5a]-1,3,5-triazine (APPT) and calmodulin-sensitive cyclic nucleotide phosphodiesterase resulted in reduction of both cyclic GMP and cyclic AMP hydrolytic activity. The inactivation was dependent upon both time of exposure to ultraviolet irradiation and the initial concentration of APPT. The photo-induced inactivation could be attenuated by the presence of cyclic GMP, 1-methyl-3-isobutylxanthine, and papaverine. alpha-Chymotrypsin treatment caused the enzyme to be fully active in the absence of calmodulin but this treatment did not alter the ability of APPT to inactivate the enzyme. Thus, inhibition of calmodulin-binding did not contribute to the photo-induced inactivation. These data indicate that APPT acts as a photoaffinity agent to covalently modify the APPT-binding site of calmodulin-sensitive phosphodiesterase.

1-Methyl-3-isobutylxanthine↗
PubMed1986

Calmodulin sensitive phosphodiesterase of porcine cerebral cortex: kinetic behavior, calmodulin activation, and stability.

The calmodulin sensitive phosphodiesterase of porcine cerebral cortex was characterized in terms of kinetic behavior, calmodulin activation, and stability. This enzyme displayed non-Michaelis-Menten kinetics in the presence or absence of calmodulin. The apparent affinity for cyclic GMP was higher than that for cyclic AMP but at saturating levels of substrate, this enzyme catalyzed the hydrolysis of cyclic AMP at a greater rate than it did cyclic GMP. The affinity of this enzyme for calmodulin was about 20-fold lower than usually reported. The apparent loss of phosphodiesterase activity after storage was found to be due to a strong association with container surfaces and could be prevented or reversed by the presence of 0.1% Triton X-100.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1986

Phorbol diesters alter the contractile responses of porcine coronary artery.

We have studied the effects of activators of the Ca++- and phospholipid-dependent enzyme protein kinase C on isometric tension development by both intact and skinned coronary artery strips. The intact strips contracted upon incubation with 12-O-tetradecanoylphorbol-13-acetate. 12-O-tetradecanoylphorbol-13-acetate produced a leftward shift in the concentration-response relationship for contraction of the tissues by K+, histamine and norepinephrine. Phorbol-12,13-dibutyrate elicited contraction of detergent-skinned artery strips when the free Ca++ concentration in the bathing media was 0.1 microM or greater. This effect was diminished greatly in the presence of polymyxin B, a putative inhibitor of protein kinase C. Phorbol-12,13-dibutyrate shifted the Ca++ concentration-tension response relationship for the skinned tissue to the left. These results are consistent with a role for protein kinase C in regulating the contractile responses of coronary arterial smooth muscle to a variety of stimuli, at least in part by increasing the sensitivity of the contractile apparatus to Ca++.

Animals↗
PubMed1985

Differences in the association of calmodulin with cyclic nucleotide phosphodiesterase in relaxed and contracted arterial strips.

Changes in the concentration of cytosolic Ca2+ are assumed to alter the activity of Ca2+-calmodulin-sensitive cyclic nucleotide phosphodiesterase in intact cells. However, this assumption is based on indirect evidence and by analogy from studies of enzyme activities in broken cell systems. We have developed a procedure for estimating the fraction of Ca2+-calmodulin-sensitive phosphodiesterase that is in an activated, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) sensitive state in intact porcine coronary artery strips. The experimental approach involves homogenization of the strips and assay of cyclic guanosine monophosphate (cyclic GMP) phosphodiesterase activity under conditions that retard changes in the amount of the complex Ca2+-calmodulin-phosphodiesterase. Our findings indicate that cyclic GMP phosphodiesterase in intact coronary artery strips does associate with Ca2+-calmodulin and that interventions that change the concentration of Ca2+ in the cytosol of the intact strip change the extent of this functional association. Exposure to histamine (10 or 100 microM) or 50 mM KCl caused contraction and an increase in EGTA-sensitive cyclic GMP phosphodiesterase activity. Isoproterenol-induced relaxation of tissues that had been caused to contract with 10 microM histamine was accompanied by a reduction in EGTA-sensitive cyclic GMP phosphodiesterase activity to the same level as that present before contraction was initiated.

3',5'-Cyclic-GMP Phosphodiesterases↗
PubMed1985

Functional properties of covalent beta-endorphin peptide/calmodulin complexes. Chlorpromazine binding and phosphodiesterase activation.

The 31-residue neuropeptide porcine beta-endorphin was shown to inhibit the Ca2+-dependent calmodulin activation of highly purified bovine brain cyclic nucleotide phosphodiesterase (3',5'-cyclic AMP 5'-nucleotidohydrolase, EC 3.1.4.17). Using a series of deletion peptides, the minimal inhibitory peptide sequence was found to correspond to beta-endorphin residues 14-25, confirming previously reported results for crude enzyme preparations. A correlation was found between the relative inhibitory potency of a particular beta-endorphin deletion peptide and the efficacy of cross-linking that peptide to calmodulin with bis(sulfosuccinimidyl) suberate, strongly implicating peptide binding to calmodulin as the mechanism of the observed inhibition. We found that relatively modest concentrations of chlorpromazine significantly reduced the efficiency of cross-linking beta-endorphin 14-31 to calmodulin. Chlorpromazine-Sepharose affinity chromatography of peptide/calmodulin adducts showed that a significant portion of the cross-linked beta-endorphin 14-31/calmodulin complex (stoichiometry of 1 mol/mol) retained the ability to interact with the immobilized phenothiazine in a Ca2+-dependent and calmodulin-displaceable manner. In contrast, the 2:1 (peptide:protein) product exhibited no affinity for the immobilized phenothiazine. The use of this affinity chromatographic step allowed preparation of homogeneous populations of both 1:1 and 2:1 beta-endorphin 13-31/calmodulin complexes and assessment of their functional characteristics. Equilibrium binding studies with chlorpromazine revealed that the covalent attachment of one peptide molecule to calmodulin perturbed all phases of Ca2+-dependent drug binding, but the adduct still bound significant quantities of chlorpromazine. The 2:1 complex, however, showed little detectable binding of the phenothiazine.(ABSTRACT TRUNCATED AT 250 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗