Carl L. Johnson

Effects of vanadate on cardiac contraction and adenylate cyclase

Abstract Vanadate produces a positive inotropic effect on ventricular muscle from rat, rabbit, guinea pig and cat; a positive inotropic effect on the atria of rat and rabbit, but a negative inotropic effect on the atria of guinea pig and cat. The effects of vanadate are completely reversible and occur in a concentration range of 10 −5 M to 10 −3 M. In this same concentration range, vanadate also causes a marked activation of cardiac adenylate cyclase suggesting that the positive inotropic action might be due in part to an elevation of cyclic AMP levels. The effects of vanadate are not influenced by alprenolol, cimetidine, or mepyramine, indicating a lack of involvement of β-adrenergic or histamine H 2 and H 1 receptors.

Differences in localization of insulin receptors and adenylate cyclase in the human placenta

The interactions of hormones with plasma membranes in the human placenta were characterized for specific fetal and maternal components. The microvillus brush border membrane, which is exposed to maternal blood in the intervillous space, was markedly enriched in specific insulin receptors but contained no hormone-sensitive adenylate cyclase. On the other hand, a basal plasma membrane fraction, which is presumably exposed more directly to fetal hormones, contained adenylate cyclase which was sensitive to prostaglandins, epinephrine, and fluoride but was not enriched in insulin receptors or other brush border markers. This study demonstrates separate fetal and maternal aspects of placental-hormone interactions. This separation and the relative impermeability of the placenta to hormones may allow for independent maternal and fetal components of the interaction of hormones with the placenta.

Schizophrenia and reduced cyclic AMP production: Evidence for the role of receptor-linked events

Reduced cyclic AMP (cAMP) production has been found in platelets of schizophrenic patients. cAMP is generated physiologically as a result of a series of steps beginning with receptor activation by a ligand, progressing through activation of the enzyme protein, adenylate cyclase. The deficit of cAMP found in the schizophrenic population may occur at any one, or at multiple steps in this cascade. The present study attempts to discriminate whether impaired adenylate cyclase itself was responsible for the cAMP deficit or whether abnormalities in receptor events or linkage are present in schizophrenics. The production of cAMP following direct stimulation of adenylate cyclase by NaF was contrasted with receptor mediated activation of adenylate cyclase by prostaglandin E1 (PGE1) in disrupted platelet preparations from schizophrenics and normal controls. cAMP formation stimulated by NaF was not different in platelets of schizophrenics as compared to controls, however, platelets of schizophrenics showed reduced response to PGE1 stimulation. The authors interpret these findings as evidence for a membrane associated abnormality of either receptor or receptor-adenylate cyclase linkage in the schizophrenias.

CALCIUM DEPENDENT REGULATION OF BRAIN AND CARDIAC MUSCLE ADENYLATE CYCLASE

The very close interdependence of Ca2+ and hormones in the overall metabolism of cyclic nucleotides has recently been emphasized by Cheung. Clearly the results presented here show that [Ca2+] in the physiological range (less than 10(-7) M to greater than 10(-6) M) has profound effects on the activity of adenylate cyclase from both brain and cardiac muscle. Whereas both brain and cardiac cyclase exhibit a Ca2+ dependent inhibition (perhaps mediated by calmodulin), only the brain cyclase is activated by Ca2+ via calmodulin. With both cyclases there is an inverse relationship between the inhibition of cyclase and the activation of calmodulin dependent (cAMP and cGMP) phosphodiesterase as a function of Ca2+ concentration. Because the IC50s for Ca2+ are the same in both heart and brain, the possibility exists that the Ca2+ inhibitory site of both cyclases is similar and perhaps identical. Considering the ability of Ca2+ to both stimulate and inhibit cyclase, one could imagine that in different species, tissues, or regions of the same tissue, there could exist multiple populations of cyclase, that is a cyclase which would only show Ca2+ dependent inhibition, Ca2+ dependent stimulation, or the biphasic response to Ca2+ (FIGURE 7). The fact that Ca2+ still regulates adenylate cyclase after various stimuli (histamine, NaF, etc.) suggests that Ca2+ may function to regulate the cyclase over shorter time periods (regardless of its state of stimulation) and that other affectors of cyclase (e.g., hormones) would serve to regulate the cyclase over longer time periods.

Studies on histamine H2 receptors coupled to cardiac adenylate cyclase Effects of guanylnucleotides and structural requirements for agonist activity

In particulate preparations from guinea-pig ventricle, histamine in the concentration range 10(-6)--10(-3) M caused a 3--5fold stimulation of adenylate cyclase activity which was dependent on the presence of GTP. The effects of fourteen analogs of histamine were examined on this cyclase preparation. Five of the compounds studied proved to be partial agonists relative to histamine while nine others had essentially the same intrinsic activity as histamine. The intrinsic activities of the partial agonists were increased by GppNHp to the extent that dimaprit, which was a partial agonist in the presence of GTP, became a full agonist in the presence of GppNHp. The relative potencies of the full agonists as activators of the cyclase were found to correlate with the relative potencies on physiologically defined H2 receptor systems. Activation of the cyclase by histamine, as well as by several of the agonist analogs, including dimaprit and tolazoline, was completely blocked by the H2 antagonist cimetidine, but was not affected by pharmacologically relevant concentrations of the H1 antagonist mepyramine, the beta-blocker alprenolol, or the alpha-blocker phentolamine. The results suggest that all the agonists studied probably interact with a common H2 receptor site on the cardiac muscle cell leading to activation of adenylate cyclase. The accompanying increase in cyclic AMP is presumably responsible for the chronotropic and inotropic effects of histamine and related compounds on cardiac muscle.

The effect of TNFa on bradykinin receptor binding, phosphatidylinositol turnover and cell growth in human A431 epidermoid carcinoma cells

In this study, we examined the effect of TNFa on bradykinin (BK) B2 receptor binding and function in human A431 epidermoid carcinoma cells. [3H]BK binds to a single class of receptors on A431 cells in a saturable and reversible manner. A binding affinity (KD) of 3.0 +/- 0.3 nM (n = 4) and a Bmax of 151 +/- 14 fmols/10(6) cells, representing approximately 90,000 BK receptors per cell, was observed. The rank order of potency for BK agonist peptides indicates that the A431 BK receptor appears to be of the B2 subtype. When A431 cells were incubated with TNFa (10 ng/ml) for 48 h prior to BK binding, a significant decrease in the number of BK receptors compared to control was observed. TNFa did not influence the affinity of BK binding to A431 cells and direct addition of TNFa to the binding assay did not effect BK binding. BK-stimulated IP1 formation appeared to be increased in TNFa treated cells compared to control whereas histamine-stimulated IP1 formation was not influenced. Both control and TNFa treated cells were greater than 95% viable. However, TNFa treated cells were blocked in the G1 phase of the cell cycle resulting in a decrease in DNA synthesis. This may be one mechanism for the TNFa-induced decrease in BK receptors in A431 cells.

Dihydropyridine receptor binding sites in the cardiomyopathic hamster heart are unchanged from control

An increase in the number of voltage dependent calcium channels has been implicated in the overload of calcium found in cardiac tissue of the cardiomyopathic hamster. We examined the binding of [3H]-(+)PN200110 to dihydropyridine receptors in cardiac muscle membranes from TO cardiomyopathic hamsters. When compared to random bred controls, there were no differences in either the Bmax or the KD for [3H]-(+)PN200110 binding using homogenates from 35 to 41-day-old TO cardiomyopathic hearts. In 8 to 9-month-old myopathic animals there were only small decreases in Bmax with no change in KD. We suggest that the calcium overload observed in cardiomyopathic hamster heart may not be due to an increased density of calcium channels as estimated by high affinity dihydropyridine receptor binding sites.

Tumor necrosis factor and interleukin-1 down-regulate receptors for substance P in human astrocytoma cells

This study examined the influence of cytokines on substance P (SP) receptors (NK1 subtype) in the human astrocytoma cell line UC11. Following trypsinization and passage, the density of SP receptors in these cells was rather low but gradually increased several fold over the course of a few days in culture. Frequent replacement of the growth medium enhanced the density of receptors even more, suggesting that growth factors in the culture medium may determine the levels of receptor. Exposure of the cells to sub-nanomolar concentrations of tumor necrosis factor (TNF alpha) or interleukin-1 beta (IL1 beta), but not interleukin-2 or interleukin-6, decreased the density of SP receptors. This was accompanied by a decrease in the ability of SP to stimulate inositolphosphate formation. The ability of histamine to activate inositolphosphate formation was not influenced by the cytokines. The decrease in SP receptor density was readily reversible on washout of the cytokines. The EC50 for TNF alpha was approximately 0.5 ng/ml, the EC50 for IL1 beta was approximately 0.1 ng/ml. Radioligand binding studies with [125I]TNF alpha indicated the presence of a low density of high affinity binding sites for this ligand: Kd = 2.5 +/- 0.6 ng/ml, Bmax = 14.8 +/- 2.7 fmol bound/mg protein (assuming trimeric form of ligand bound). The most likely explanation for the cytokine effect is an inhibition of the synthesis of new receptors.

Inhibition of human skin fibroblast proliferation by histamine and phorbol esters is mediated by protein kinase C

The proliferation of human skin fibroblasts in culture was examined using a [3H]thymidine incorporation assay. Histamine inhibited thymidine incorporation with an IC50 of about 0.2 microM. This effect was blocked by the H1 receptor antagonist mepyramine but not by the H2 receptor antagonist cimetidine. Protein kinase C activators, including several phorbol esters and mezerine, also inhibited thymidine incorporation. The IC50 for beta-phorbol 12,13-didecanoate was less than 0.1 nM. The alpha-isomer of this compound was inactive. Long-term treatment of cells with the beta-isomer eliminated the ability of both histamine and phorbol ester to inhibit thymidine incorporation, presumably due to downregulation of protein kinase C. Our results suggest that histamine H1 receptors are linked to activation of protein kinase C and that activation of this enzyme leads to an inhibition of cell proliferation.

Effects of dimaprit on growth and differentiation of human promyelocytic cell line, HL-60

The human promyelocytic cell line HL-60, differentiates in response to a variety of agents including dibutyryl cAMP and agents which increase intracellular cAMP concentrations (phosphodiesterase inhibitors, PGE2, and cholera toxin). HL-60 is also known to be rich in H2 -histamine sensitive adenylate cyclase activity. The present study was therefore designed to test the effects of H2-stimulation on growth and differentiation of HL-60 using the potent H2 agonist dimaprit. Dimaprit markedly increased cAMP production in a dose-dependent manner reaching maximal levels after 30-60 minutes. Intracellular cAMP levels decreased thereafter and by 24 hours were approximately 2-3 fold increased above control. Intracellular cAMP levels were not altered by dimaprit (10(-7)M to 10(-4)M) at 4 days in culture compared to either untreated HL-60 cells or dimethylsulfoxide (DMSO) (1.3%) treated cells. While exponential growth was unaltered by dimaprit (10(-7)M to 10(-4)M) as compared to control, dimaprit induced i) morphologic maturation to the myelocyte and metamyelocyte form with no differentiation seen beyond the metamyelocyte even after 6 days in culture, ii) increased NBT reductase activity and iii) dose-dependent increase in lysozyme activity which could be completely blocked by cimetidine, a specific H2 antagonist. Dimaprit-induced differentiation of HL-60 cells was associated with an initial but transient increase in intracellular cAMP production. Maturation beyond the metamyelocyte stage was not observed. Acquisition of NBT reductase and lysozyme activity correlated with morphologic maturation.