Search PubMed⌕ Search

Biomedical subjects

J G Hardman

Publications and source records attributed to J G Hardman.

At least 55 records · Page 3Linked to original sources
PubMed1978

Characteristics of adenylate cyclase activity in pig coronary arteries.

Characteristics of adenylate cyclase activity of the intima plus media layer of pig coronary arteries have been studied. Enzyme activity in the 10,000 xg particulate fraction was linear with respect to time and protein during short incubations. Linear kinetics were observed for MgATP as a substrate and free Mg2+ as an activator. Mn2+ added in excess of ATP concentrations was only slightly stimulatory or inhibitory. Adenosine, GTP and GPP(N)P inhibited enzyme activity. Adenosine decreased the Vmax and the Ka for Mg2+ but had no effect on the Km for MgATP. The inhibition by adenosine was reversible, while that by GPP(N)P appeared to be irreversible. Although preincubation of the enzyme with GPP(N)P for short times caused irreversible inhibition, preincubation for more than 15 min caused up to 3-fold activation. Isoproterenol added to the incubation produced no or very slight stimulation with or without GPP(N)P or GTP, and preincubation with isoproterenol alone caused irreversible inhibition. However, preincubation with isoproterenol plus GPP(N)P for 30 min caused irreversible activation exceeding that observed with GPP(N)P alone. These observations suggest that vascular adenylate cyclase, like the enzyme from the other tissues, can exist in multiple states of activity and responsiveness.

Adenosine↗
PubMed1976

Implications of cross inhibitory interactions of potential mediators of hormone and neurotransmitter action.

Mediators of hormone and neurotransmitter action may exert negative control on the accumulation of one another (adenosine 3':5'-cyclic monophosphate, or conversely). A model in which one agonist stimulates the formation of two mediators which inhibit each other's accumulation has been simulated. Three types of agonist-receptor interaction, five mechanisms of inhibition, and three types of basal activity have been considered which lead to a set of 45 descriptions of the general cross inhibition model. For these submodels, pattern of the relation of agonist to mediator concentrations at steady state conditions have been defined. Some patterns are complex with mediator curves exhibiting extrema, one a maximum and the other a minimum. The complexity of the pattern depends on the submodel and on the degree of asymmetry between the parameters of each of the mediator pathways (e.g.,affinities for the agonist, strength of the inhibitions, etc.). This simple model can thus account for complex experimental results without requiring the postulation of elaborate molecular models of agonist-receptor interaction. The simulations presented emphasize the necessity of investigating a wide range of agonist concentrations.

Calcium↗
PubMed1976

Effects of egg factors on cyclic nucleotide metabolism in sea urchin sperm.

Cyclic AMP in Strongylocentrotus purpuratus sperm was elevated approximately 2-fold by theophylline or 1-methyl-3-isobutylxanthine. Factors released from sea urchin eggs (FRE) elevated sperm cyclic AMP by about 7-fold within 1 min, and the combination of FRE with theophylline increased sperm cyclic AMP up to 100-fold within 1 min. Cyclic GMP in sea urchin sperm was slightly elevated by theophylline, but was lowered by FRE. Cyclic GMP in sperm treated with FRE plus theophylline was not higher than in sperm treated with theophylline alone. The ability of FRE-containing sea water to increase sperm cyclic AMP in the presence of theophylline was altered only slightly if at all by boiling, but it was decreased by about 50% by dialysis and destroyed by ashing. Filtration of FRE on Sephadex G-50 columns yielded two peaks of cyclic AMP-elevating activity. One peak (peak I) was eluted at the column void volume, and the other (peak II) was retained by the column. The cyclic GMP-lowering activity was located in fractions approximately corresponding to peak I of cyclic AMP-elevating activity. Dialysis of FRE-containing sea water before its application to the G-50 column virtually eliminated peak II of the cyclic AMP-elevating activity. When the cyclic AMP-elevating activity in peak I was filtered on Bio Gel A-5m columns, it also migrated at or near the column void volume. Fractions corresponding to peak I contained material that inhibited both guanylate and adenylate cyclase activities in broken cell preparations of sperm and guanylate cyclase from rat lung. The inhibitory material was stable to boiling, non-dialyzable, and destroyed by ashing. Under a variety of conditions, FRE-containing sea water or cyclic AMP-elevating peaks I or II did not stimulate sperm adenylate cyclase activity in broken cell preparations.

Adenylyl Cyclases↗
PubMed1975

Effects of metals and nucleotides on the inactivation of sea urchin sperm guanylate cyclase by heat and N-ethylmaleimide.

Preincubation of sea urchin sperm guanylate cyclase at 35, 37, 40, or 43 degrees resultedin inactivation. Various metals were able to protect guanylate cyclase against heat inactivation. Estimated binary enzyme-metal dissociation constants for Mn2+, Fe2+, La3+, Ca2+, Ba2+, Mg2+, Co2+, and Ni2+ were 123, 361, 5.5, 692, 984, 335, 79, and 47 muM, respectively. Extrapolated rates of enzyme denaturation in the presence of saturating concentrations of metal divided by the rates of enzyme denaturation in the absence of metal gave values of 0.13, 0.08, minus 0.1, 0.30, 0.59, 0.66, 0.28, and 0.42 for Mn2+, Fe2+, La3+, Ca2+, Ba2+, Mg2+, Co2+, and Ni2+, respectively. GTP, MgGTP, and SrGTP protected the enzyme only slightly against heat inactivation, but CaGTP and MnGTP protected substantially. Neither CaGTP nor MnGTP protected maximally, however, unless the metal concentration exceeded that of GTP. At fixed free Mn2+ or free Ca2+ concentrations, protection curves as a function of MnGTP or CaGTP appeared to be sigmoidal, suggesting multiple nucleotide binding sites. MnATP also protected against heat, but CaATP was virtually ineffective. Sea urchin sperm guanylate cyclase was inactivated by N-ethylmaleimide; CaGTP and MnATP were effective protectants with estimated binary enzyme-Me2+ nucleoside triphosphate dissociation constants of 40 and 170 muM, respectively. MnGTP protected only slightly or not at all against N-ethylmaleimide. These results suggest that: (a) sea urchin sperm guanylate cyclase binds free metal, (b) the binding of free metal is required for protection by nucleotides, and (c) the enzyme contains multiple nucleotide binding sites.

Adenosine Triphosphate↗
PubMed1975

Hydrolysis of guanosine and adenosine 3',5'-monophosphates by rat blood.

Cyclic nucleotide phosphodiesterase activity was measured in whole blood, plasma, and suspensions of platelets and erythrocytes from rats. In fresh whole blood, apparent phosphodiesterase activity was low, but it rose strikingly during the hour after blood withdrawal. The apparent phosphodiesterase activity in platelet-free plasma showed no such increase, but that in platelet-enriched plasma increased in parallel with that in whole blood. The apparent phosphodiesterase activity of blood or of platelet-enriched plasma also was increased markedly by sonication. The increase in rat blood phosphodiesterase activity with aging thus appeared to be due to damage of platelets. Most of the phosphodiesterase activity in rat erythrocytes and platelets was located in the soluble fraction of sonicated preparations, but the total enzyme activities from the two sources exhibited marked differences in substrate specificity. With erythrocyte preparations, the rate of hydrolysis of muM concentrations of cyclic AMP was approx. 50 times that of cyclic GMP, while with platelet preparations, cyclic GMP was hydrolyzed about 20 times faster than cyclic AMP at muM levels. The activity of phosphodiesterase in platelets was much greater than that in erythrocytes at all concentrations of both substrates.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1975

Characterization of particulate and soluble guanylate cyclases from rat lung.

Rat lung homogenates contained significant amounts of guanylate cyclase activity in both 100,000 times g (60 min) particulate and supernatant fractions. In the presence of detergent, the particulate fraction contained 40% as much activity as did the supernatant fraction. Detergent-dispersed particulate and partially purified soluble guanylate cyclase preparations were characterized with respect to divalent cation requirements, divalent cation interactions, kinetic behavior, and gel filtration profiles. Both soluble and particulate guanylate cyclases required divalent cation for activity. The soluble preparation was 10 times more active in the presence of Mn-2plus than in the presence of Mg-2plus or Ca-2plus and no detectable activity was seen with Ba-2plus or Sr-2plus. Particulate guanylate cyclase activity was detectable only in the presence of Mn-2plus. Both enzyme preparations required Mn-2plus in excess of GTP for optimal activity at subsaturating amounts of GTP. At near-saturating GTP, the soluble enzyme required excess Mn-2plus, but the particulate enzyme did not. For kinetic analyses the enzymes were considered to require two substrates: metal-GTP and Me-2plus. Apparent negative cooperative behavior was seen with the soluble enzyme when excess Mn-2plus (in excess of GTP) was varied from 0.01 to 0.2 mM; above 0.2 mM excess Mn-2plus classical kinetic behavior was seen with an apparent KMn-2plus of 0.2 mM at near-saturating MnGTP. Similar studies using the particulate preparation yielded only classical kinetic behavior, but the apparent KMn-2plus decreased to near zero when MnGTP was near-saturating. Kinetic patterns for the particulate and soluble enzymes also differed when reciprocal initial velocities were plotted as a function of reciprocal MnGTP concentrations; classical kinetic behavior was seen with the soluble enzyme with an apparent KMnGTP of about 12 muM (at near-saturating excess Mn-2plus), whereas apparent positive cooperative behavior was seen with the particulate preparation (Hill coefficient equals 1.6, S0.5 EQUALS 70 MUM. Ca-2plus "activation" of soluble guanylate cyclase was related to the Mn-2plus:GTP ratio. Activation was most apparent when saturating amounts of Mn-2plus and MnGTP. At relatively high concentrations of Ca-2plus (0.1 to 4 mM), the addition of 10 muM Mn-2plus resulted in a 3- to 5-fold increase in soluble guanylate cyclase activity. In contrast, Ca-2plus sharply inhibited particulate guanylate cyclase activity. Gel filtration profiles of particulate and soluble preparations indicated differences in physical properties of the enzymes. As estimated by gel filtration, particulate (detergent-dispersed)evels. Here, removal of renal tissue is contraindicated. In all renal hy

Animals↗
PubMed1975

Effects of cations on guanylate cyclase of sea urchin sperm.

Mn2+ and to some degree Fe2+, but not Mg+, Ca2+, ba2+, Sr2+, Co2+, Ni2+, La3+, or Fe3+ were able to serve as effective metal cofactors for sea urchin sperm guanylate cyclase. The apparent Michaelis constant for Mn2+ in the presence of 0.25 mM MnGTP was 0.23 mM. In the presence of a fixed free mn2+ concentration, variation in mngTP resulted in sigmoid velocity-substrate plots and in reciprocal plots that were concave upward. These positive cooperative patterns were observed at both pH 7.0 and 7.8 and in the presence or absence of Triton X-100. When Mn2+ and GTP were equimolar, Ca2+, Ba2+, Sr2+, and Mg2+ increased apparent guanylate cyclase activity. This increase in enzyme activity at least could be accounted for partially by an increase in free Mn2+ concentration caused by the complex formation of GTP with the added metals. However, even at relatively low GTP concentrations and with Mn2+ concentrations in excess of GTP, Ca2+, Sr2+, and Ba2+ significantly increased guanosine 3':5'-monophosphate production. As the total GTP concentration was increased, the degree of stimulation in the presence of Ca2+ decreased, despite maintenance of a fixed total concentration of Ca2+ and a fixed free concentration of Mn2+, suggesting that the concentration of CaGTP and MnGTP were determining factors in the observed response. The concave upward reciprocal plots of velocity against MnGTP concentration were changed to linear plots in the presence of CaGTP or SrGTP. These results suggest that sea urchin sperm guanylate cyclase contains multiple nucleotide binding sites and that stimulation of guanosine 3':5'-monophosphate synthesis by Ca2+, Sr2+, and perhaps other metals may reflect interaction of a metal-GTP complex with enzyme as either an effector or a substrate.

Animals↗
PubMed1975

Enzymatic formation of inosine 3',5'-monophosphate and of 2'-deoxyguanosine 3',5'-monophosphate. Inosinate and deoxyguanylate cyclase activity.

Enzymes in particulate fractions from sea urchin sperm and in soluble fractions from rat lung were shown to catalyze the formation of inosine 3',5'-monophosphate (cyclic IMP) and of 2'-deoxyguanosine 3',5'-monophosphate (cyclic dGMP) from ITP and dGTP, respectively. With sea urchin sperm particulate fractions, Mn2+ was an essential metal cofactor for inosinate, deoxyguanylate, guanylate and adenylate cyclase activities. Heat-inactivation studies differentiated inosinate and deoxyguanylate cyclase activities from adenylate cyclase, but indicated an association of these activities with guanylate cyclase. Preincubation of sea urchin sperm particulate fractions with trypsin altered in a very similar manner guanylate, inosinate, and deoxyguanylate cyclase activities, and various metals and metal-nucleotide combinations protected the three cyclase activities to comparable degrees against trypsin. The relative guanylate, deoxyguanylate and inosinate cyclase activities at 0.1 mM nucleoside triphosphate were 1.0, 0.5 and 0.08, respectively. With these three cyclase activities, plots of reciprocal velocities against reciprocal Mn2+-nucleoside triphosphate concentrations were concave upward, suggesting positive homotropic effects. With rat lung soluble preparations, relative guanylate, deoxyguanylate, inosinate and adenylate cyclase activities at 0.09 mM nucleoside triphosphate were 1.0, 1.7, 0.1 and 0, respectively. MnGTP was a competitive inhibitor of deoxyguanylate cyclase activity (Ki equals 12.2 muM) and MndGTP was a competitive inhibitor of guanylate cyclase activity (Ki equals 16.2 muM). Inhibition studies using ITP were not conducted. When soluble fractions from rat lung were applied to Bio-Gel A 1.5 m columns, elution profiles of guanylate, deoxyguanylate and inosinate cyclase activities were similar. These results suggest that deoxyguanylate, guanylate and inosinate cyclase activities reside within the same protein molecule.

Adenosine Triphosphate↗
PubMed1975

Effects of norepinephrine on cyclic nucleotide levels in the ductus deferens of the rat.

The effect of 1-norepinephrine on cyclic nucleotide levels was studied in rat ductus deferens. Norepinephrine (0.01-0.3 mM) increased cyclic AMP and cyclic GMP levels in a concentration-dependent manner. Cyclic GMP was increased by about two-fold and cyclic AMP by 30%-40% by 0.1 mM norepinephrine after 3 min. Phenylephrine (0.1 mM) increased cyclic GMP levels about twofold without affecting cyclic AMP. Cyclic GMP was maximally increased by norepinephrine or phenylephrine after 3-10 min, but was not significantly changed after 20 sec, when contraction was fully developed. Atropine did not affect the norepinephrine-induced changes in cyclic nucleotide levels; phentolamine blocked the effect of norepinephrine on cyclic GMP, and propanolol blocked that on cyclic AMP. These findings indicate that in the ductus deferens alpha-adrenergic receptors are involved in the effect of catecholamines on cyclic GMP, and beta-adrenergic receptors are involved in the effect on cyclic AMP levels. The presence of Ca-2+ was required for the effect of norepinephrine on cyclic GMP, but not on cyclic AMP.

Acetylcholine↗
PubMed1975

Effect of hypoglycemia on extracellular levels of cyclic AMP in man.

This study was designed to assess the response of cyclic AMP to "endogenous" hormonal stimulation resulting from insulin-induced hypoglycemia. Insulin was administered to four normal subjects and four adrenalectomized patients. Hypoglycemia resulted in four-fold increases in plasma cyclic AMP. This response is thought to be secondary to beta-adrenergic stimulation for the following reasons: (1) the response was absent in adrenalectomized, cortisol-treated subjects; (2) it was abolished by propranolol; and (3) urinary excretion of cyclic AMP did not reflect the rise in plasma cyclic AMP.

Adrenal Glands↗
PubMed1975

Studies of the elevated extracellular concentration of cyclic AMP in uremic man.

This study was designed to elucidate the mechanism of elevation of plasma cyclic AMP in uremic man. Plasma cyclic AMP was measured in 15 normal subjects and in 18 patients with severe renal failure. In some members from both groups the kinetic parameters of the metabolism of extracellular cyclic AMP were measured. Plasma cyclic AMP was elevated from 23 nM in control subjects to 59 nM in uremic patients, regardless of the presence or absence of the kidneys or parathyroid glands. A single pass of uremic blood through a Kiil hemodialyzer decreased plasma cyclic AMP from 58 to 30 nM. The clearance of cyclic AMP by the dialyzer correlated directly with the blood flow passing through the machine. Hemodialysis for 6 h decreased plasma cyclic AMP levels in the systemic circulation by only 12%. Studies with tritiated cyclic AMP revealed a plasma clearance rate of 624 ml/min in normal subjects and of 344 ml/min in patients with uremia. Such a large decrease in plasma clearance rate cannot be explained by a failure of urinary excretion of cyclic AMP and suggests impairment of "metabolic clearance." In addition, the "plasms production rate" of cyclic AMP was 65% higher in patients with renal failure than in normal subjects. It is concluded that the elevation of plasma cyclic AMP in uremic man is due to a combination of: (a) lack of urinary excretion, (b) decreases metabolic clearance, and (c) increased production of plasma cyclic AMP.

Adult↗
PubMed1975

Regulation of cyclic GMP levels in the ductus deferens of the rat.

The cyclic GMP level in the ductus deferens is elevated by acetylcholine, norepinephrine, KCl, and the phosphodiesterase inhibitor SC-2964. The presence of extracellular Ca++ is required for the effects of all of these agents on cyclic GMP levels. In addition, Ca++ appears to be an important factor for the basal turnover of cyclic GMP in this tissue, but it may be less important in other tissues. These observations have led us to the following working hypothesis (Fig. 5): The interactions of some hormones or neurotransmitters with membrane receptors secondarily increase cyclic GMP formation after primarily increasing the influx of extracellular Ca++ or changing the distribution of Ca++ among intracellular pools or compartments. However, in addition to this possibility, other hormonal effects on particulate and/or soluble guanylate cyclase that do not involve Ca++ mediation must also be considered. Some agents that are known to increase cyclic GMP in tissues have been reported in preliminary communications to activate cell-free preparations of guanylate cyclase (Amer and McKINNEY, 1973; White, Ignarro, and George, 1973), but these reports have not yet been confirmed by other laboratories. Secretin has been reported to stimulate guanylate cyclase activity from several tissues (Thompson, Johnson, Lavis, and Williams, 1974), but the significance of this report is unclear since secretin has not yet been shown to increase cyclic GMP levels in any tissue. Thus, although not convincingly established, some hormones may increase particulate guanylate cyclase activity in a manner similar to that by which hormones increase adenylate cyclase activity. Alternatively, some hormones may increase soluble guanylate cyclase activity with mediating factors other than Ca++ being involved, or hormone-receptor interaction at the plasma membrane could conceivably induce a dislocation and change in effective activity of a reversibly bound, membrane-associated guanylate cyclase. Elucidating which or how many of these possibilities are operative will require thorough study and understanding of the fundamental behavior and properties of soluble and particulate guanylate cyclase activities.

Acetylcholine↗