Effects of heptanol on the neurogenic and myogenic contractions of the guinea-pig vas deferens. (1/824)

1. The effects of the putative gap junction uncoupler, 1-heptanol, on the neurogenic and myogenic contractile responses of guinea-pig vas deferens were studied in vitro. 2. Superfusion of 2.0 mM heptanol for 20-30 min produced the following reversible changes in the biphasic neurogenic contractile response (8 trials): (i) suppression of both phases; (ii) delayed development of both the first as well as the second phase, accompanied by complete temporal separation of the two phases; (iii) prominent oscillations of force during the second (noradrenergic) phase only. 3. To eliminate prejunctional effects of heptanol, myogenic contractions were evoked by field stimulation of the vas in the presence of suramin (200 microM) and prazosin (1 microM). Heptanol (2.0 mM) abolished these contractions reversibly. 4. These results show that (i) heptanol inhibits both excitatory junction potential (EJP)-dependent and non EJP-dependent contractions of the vas; (ii) a postjunctional site of action of heptanol, probably intercellular uncoupling of smooth muscle cells, contributes to the inhibition of contraction.  (+info)

Suramin and suramin analogs activate skeletal muscle ryanodine receptor via a calmodulin binding site. (2/824)

Contraction of skeletal muscle is triggered by the rapid release of Ca2+ from the sarcoplasmic reticulum via the ryanodine receptor/calcium-release channel. The trypanocidal drug suramin is an efficient activator of the ryanodine receptor. Here, we used high-affinity [3H]ryanodine binding to sarcoplasmic reticulum from rabbit skeletal muscle to screen for more potent analogs of suramin. This approach resulted in the identification of NF307, which accelerates the association rate of [3H]ryanodine binding with an EC50 = 91 +/- 7 microM at 0.19 microM calculated free Ca2+. In single-channel recordings with the purified ryanodine receptor, NF307 increased mean open probability at 0.6 microM Ca2+ from 0.020 +/- 0.006 to 0.53 +/- 0.07 with no effect on current amplitude and unitary conductance. Like caffeine, NF307 exerts a very pronounced Ca2+-sensitizing effect (EC50 of Ca2+ shifted approximately 10-fold by saturating NF307 concentrations). Conversely, increasing concentrations of free Ca2+ sensitized the receptor for NF307 (EC50 = 14.6 +/- 3.5 microM at 0.82 microM estimated free Ca2+). The effects of NF307 and caffeine on [3H]ryanodine binding were additive, irrespective of the Ca2+ concentration. In contrast, the effects of calmodulin, which activates and inhibits the ryanodine receptor in the absence and presence of Ca2+, respectively, and of NF307 were mutually antagonistic. If the purified ryanodine receptor was prebound to a calmodulin-Sepharose matrix, 100 microM NF307 and 300 microM suramin eluted the purified ryanodine receptor to an extent that was comparable to the effect of 10 microM calmodulin. We conclude that NF307 and suramin interact directly with a calmodulin binding domain of the ryanodine receptor. Because of its potent calcium-sensitizing effect, NF307 may represent a lead compound in the search of synthetic ryanodine receptor ligands.  (+info)

Regulation of gelatinase B production in corneal cells is independent of autocrine IL-1alpha. (3/824)

PURPOSE: The matrix metalloproteinase gelatinase B is synthesized by cells at the leading edge of the corneal epithelium migrating to heal a wound. Recent data from the authors' laboratory suggest that excessive synthesis contributes to repair defects. The goal of the study reported here was to investigate mechanisms controlling gelatinase B production by corneal epithelial cells. METHODS: Freshly isolated cultures of corneal epithelial cells and early passage stromal fibroblasts from rabbit were used for these studies. RESULTS: In a previous study, it was found that the cytokine interleukin (IL)-1alpha is released into the culture medium of corneal epithelial cells more efficiently when they are plated at low density with limited cell-cell contact than when plated at high density. In this study, we show that production of gelatinase B by these cells is similarly affected by cell plating density. However, it is further demonstrated that these two events are not dependent on one another but occur in parallel: IL-1alpha does not regulate gelatinase B production (synthesis), nor was there evidence that any other secreted autocrine cytokine acts as mediator. Instead, our data suggest that gelatinase B production is downregulated directly by high cell density and indicate a connection to the level of protein kinase C activity. Nevertheless, the anticancer agent suramin, which blocks collagenase synthesis by interfering with autocrine cytokine-receptor interactions, still inhibits synthesis of gelatinase B. CONCLUSIONS: Unlike collagenase synthesis by corneal stromal fibroblasts, production (synthesis) of gelatinase B does not appear to be controlled by secreted autocrine cytokines but can still be inhibited by suramin. Suramin may make an effective therapeutic agent for controlling pathologic overproduction of gelatinase B in corneal ulcers.  (+info)

Local regulation of vasopressin and oxytocin secretion by extracellular ATP in the isolated posterior lobe of the rat hypophysis. (4/824)

It is now widely accepted that ATP functions as a signalling substance in the nervous system. The presence of P2 receptors mediating the action of extracellular ATP in brain regions involved in hormonal regulation raises the possibility that a similar role for ATP might also exist in the neuroendocrine system. In this study, the release from the rat isolated neurohypophysis preparation of endogenous ATP, oxytocin and vasopressin (AVP) were measured simultaneously using luciferin-luciferase and RIA techniques. After 70 min preperfusion, electrical field stimulation caused a rapid increase in the amount of ATP in the effluent and the release of AVP and oxytocin also increased stimulation-dependently. Inhibition of voltage-dependent Na+ channels by tetrodotoxin (1 microM) reduced the stimulation-evoked release of AVP and oxytocin; however, the evoked release of ATP remained unaffected. The effect of endogenous ATP on the hormone secretion was tested by suramin (300 microM), the P2 receptor antagonist. Suramin significantly increased the release of AVP, and the release of oxytocin was also enhanced. ATP, when applied to the superfusing medium, decreased the release of AVP, but not that of oxytocin, and its effect was prevented by suramin. ATP (60 nmol), added to the tissues, was readily decomposed to ADP, AMP and adenosine measured by HPLC combined with ultraviolet light detection, and the kinetic parameters of the enzymes responsible for inactivation of ATP (ectoATPase and ecto5'-nucleotidase) were also determined (Km=264+/-2.7 and 334+/-165 microM and vmax=6.7+/-1.1 and 2.54+/-0.24 nmol/min per preparation (n=3) for ectoATPase and ecto5'-nucleotidase respectively). Taken together, our data demonstrate the stimulation-dependent release, P2 receptor-mediated action and extracellular metabolism of endogenous ATP in the posterior lobe of the hypophysis and indicate its role, as a paracrine regulator, in the local control of hormone secretion.  (+info)

Antioxidants improve impaired insulin-mediated glucose uptake and prevent migration and proliferation of cultured rabbit coronary smooth muscle cells induced by high glucose. (5/824)

BACKGROUND: To explore the role of intracellular oxidative stress in high glucose-induced atherogenesis, we examined the effect of probucol and/or alpha-tocopherol on the migration and growth characteristics of cultured rabbit coronary vascular smooth muscle cells (VSMCs). METHODS AND RESULTS: Chronic high-glucose-medium (22. 2 mmol/L) treatment increased platelet-derived growth factor (PDGF)-BB-mediated VSMC migration, [3H]thymidine incorporation, and cell number compared with VSMCs treated with normal-glucose medium (5.6 mmol/L+16.6 mmol/L mannose). Probucol and alpha-tocopherol significantly suppressed high glucose-induced increase in VSMC migration, cell number, and [3H]thymidine incorporation. Probucol and alpha-tocopherol suppressed high glucose-induced elevation of the cytosolic ratio of NADH/NAD+, phospholipase D, and membrane-bound protein kinase C activation. Probucol, alpha-tocopherol, and calphostin C improved the high glucose-induced suppression of insulin-mediated [3H]deoxyglucose uptake. Chronic high-glucose treatment increased the oxidative stress, which was significantly suppressed by probucol, alpha-tocopherol, suramin, and calphostin C. CONCLUSIONS: These findings suggest that probucol and alpha-tocopherol may suppress high glucose-induced VSMC migration and proliferation via suppression of increases in the cytosolic ratio of free NADH/NAD+, phospholipase D, and protein kinase C activation induced by high glucose, which result in reduction in intracellular oxidative stress.  (+info)

Cell surface retention sequence binding protein-1 interacts with the v-sis gene product and platelet-derived growth factor beta-type receptor in simian sarcoma virus-transformed cells. (6/824)

The cell surface retention sequence (CRS) binding protein-1 (CRSBP-1) is a newly identified membrane glycoprotein which is hypothesized to be responsible for cell surface retention of the oncogene v-sis and c-sis gene products and other secretory proteins containing CRSs. In simian sarcoma virus-transformed NIH 3T3 cells (SSV-NIH 3T3 cells), a fraction of CRSBP-1 was demonstrated at the cell surface and underwent internalization/recycling as revealed by cell surface 125I labeling and its resistance/sensitivity to trypsin digestion. However, the majority of CRSBP-1 was localized in intracellular compartments as evidenced by the resistance of most of the 35S-metabolically labeled CRSBP-1 to trypsin digestion, and by indirect immunofluorescent staining. CRSBP-1 appeared to form complexes with proteolytically processed forms (generated at and/or after the trans-Golgi network) of the v-sis gene product and with a approximately 140-kDa proteolytically cleaved form of the platelet-derived growth factor (PDGF) beta-type receptor, as demonstrated by metabolic labeling and co-immunoprecipitation. CRSBP-1, like the v-sis gene product and PDGF beta-type receptor, underwent rapid turnover which was blocked in the presence of 100 microM suramin. In normal and other transformed NIH 3T3 cells, CRSBP-1 was relatively stable and did not undergo rapid turnover and internalization/recycling at the cell surface. These results suggest that in SSV-NIH 3T3 cells, CRSBP-1 interacts with and forms ternary and binary complexes with the newly synthesized v-sis gene product and PDGF beta-type receptor at the trans-Golgi network and that the stable binary (CRSBP-1.v-sis gene product) complex is transported to the cell surface where it presents the v-sis gene product to unoccupied PDGF beta-type receptors during internalization/recycling.  (+info)

Modulation of ATP-responses at recombinant rP2X4 receptors by extracellular pH and zinc. (7/824)

The modulatory effects of extracellular H+ and Zn2+ were tested against ATP-responses at rat P2X4 (rP2X4) receptors expressed in Xenopus oocytes under voltage-clamp conditions. ATP (0.1-100 microM, at pH 7.5), evoked inward currents via rP2X4 receptors (EC50 value, 4.1+/-0.98 microM; nH, 1.2+/-0.1). ATP potency was reduced 2 fold, at pH 6.5, without altering maximal activity. ATP potency was reduced by a further 4 fold, at pH 5.5, and the maximal activity of ATP was also reduced. Alkaline conditions (pH 8.0) had no effect on ATP-responses. Zn2+ (100 nM - 10 microM) potentiated ATP-responses at the rP2X4 receptor by 2 fold, whereas higher concentrations (30 microM - 1 mM) inhibited ATP-responses. Zn2+ potentiation was due to an increase in ATP potency, whereas its inhibitory action was due to a reduction in ATP efficacy. Zn2+ modulation of ATP-responses was pH-dependent. At pH 6.5, the bell-shaped curve for Zn2+ was shifted to the right by 1 log unit. At pH 5.5, Zn2+ potentiation was abolished and its inhibitory effect reduced considerably. Suramin (50 microM) also potentiated ATP-responses at rP2X4 receptors. Neither H+ (pH 6.5 and 5.5), Zn2+ (10-100 microM) or a combination of both failed to reveal an inhibitory action of suramin at rP2X4 receptors. In conclusion, H+ and Zn2+ exerted opposite effects on the rP2X4 receptor by lowering and raising agonist potency, respectively. H+ (> or = 3 microM) and Zn2+ (> or = 30 microM) also reduces agonist efficacy by lowering the number of rP2X4 receptors available for activation. The striking differences between the modulatory actions of H+ and Zn2+ at rP2X4 and rP2X2 receptors are discussed.  (+info)

Neural modulation of the cyclic electrical and mechanical activity in the rat colonic circular muscle: putative role of ATP and NO. (8/824)

1. The rat colonic circular muscle displays cyclic episodes of myenteric potential oscillations (MPOs), each of them associated with a spontaneous contraction. Nifedipine 1 microM abolished both MPOs and their associated contractions. TTX (1 microM) increased the amplitude and frequency of spontaneous contractions. 2. Electrical field stimulation (EFS) induced a non-adrenergic non-cholinergic (NANC) inhibitory junction potential (IJP), with two phases: an initial fast hyperpolarization (characterized by IJP amplitude) and a sustained hyperpolarization (characterized by IJP duration). 3. Sodium nitroprusside (10 microM) hyperpolarized and abolished spontaneous contractions even in presence of TTX or 1 microM apamin. ATP (100 microM) also hyperpolarized and abolished spontaneous contractions but its effects were decreased by TTX and abolished by apamin. 4. Suramin (100 microM) or apamin reduced the amplitude of the IJPs, but did not affect their duration. Incubation with L-NOARG (1 mM) reduced the duration but not the amplitude of the IJPs. In presence of L-NOARG plus suramin or L-NOARG plus apamin, both duration and amplitude of the IJPs were reduced but a residual IJP could still be recorded. 5. We conclude that the mechanical and electrical cyclic activity of the rat colonic circular muscle is modulated but not originated by the enteric nervous system and involves L-type calcium channel activity. EFS induces release of NANC inhibitory neurotransmitters which hyperpolarize and relax smooth muscle cells. Both ATP and NO are involved in IJP generation: ATP is responsible for the first phase of the IJPs involving activation of apamin-sensitive potassium channels, whereas NO initiates the second phase which is independent of the activation of such channels.  (+info)

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