Effects of heptanol on the neurogenic and myogenic contractions of the guinea-pig vas deferens.
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)
Further evidence for the selective disruption of intercellular communication by heptanol.
The lack of selective gap junctional uncoupling agents has hampered evaluation of the contribution of intercellular communication to pharmacomechanical coupling and vascular contractility. Thus we further explored the utility and selectivity of heptanol as a gap junctional uncoupling agent in isolated rat aortic rings. Fifty-two aortic rings were obtained from 15 rats and were precontracted to approximately 75% of maximum with phenylephrine (PE). When contraction achieved steady state (approximately 5 min), a single concentration of heptanol (200 microM) was added to each aortic ring at 1- to 3-min intervals for up to 42 min post-PE addition. At early time points (5-10 min after PE), heptanol elicited an approximately 50% loss of tension (i.e., relaxation). At subsequent time points post-PE, a gradual and time-dependent decrease in the magnitude of the heptanol-induced relaxation was observed until, after approximately 40 min, addition of heptanol was associated with little, if any, detectable relaxation. Linear regression analysis of the magnitude of the heptanol-induced relaxation vs. the square root of the elapsed time interval (from addition of PE) revealed a highly significant negative correlation (P < 0.001, R = 0.81). Studies conducted on KCl-precontracted aortic rings revealed no detectable heptanol-induced relaxation after development of the steady-state KCl-induced contraction. These data extend our previous observations to further document the potential utility of heptanol as a "relatively selective" uncoupling agent. (+info)
Quantal evoked depolarizations underlying the excitatory junction potential of the guinea-pig isolated vas deferens.
1. The effects of a putative gap junction uncoupling agent, heptanol, on the intracellularly recorded junction potentials of the guinea-pig isolated vas deferens have been investigated. 2. After the stimulation-evoked excitatory junction potentials (EJPs) had been suppressed by heptanol (2.0 mM) to undetectable levels, a different pattern of evoked activity ensued. This consisted of transient depolarizations that were similar to EJPs in being stimulus locked and in occurring at a fixed latency, but differed from EJPs in that they occurred intermittently and had considerably briefer time courses. 3. Analysis of the amplitudes and temporal parameters of the rapid residual depolarizations revealed a close similarity with spontaneous EJPs (SEJPs). There was no statistically significant difference between the rise times, time constants of decay and durations of the rapid residual depolarizations and of SEJPs. 4. Selected evoked depolarizations were virtually identical to SEJPs occurring in the same cell. Evoked depolarizations of closely similar amplitude and time course also occurred, usually within a few stimuli of each other. 5. These depolarizations appear to represent the individual quantal depolarizations that normally underlie the EJP and are therefore termed 'quantal excitatory junction potentials' (QEJPs) to distinguish them from both EJPs and SEJPs. 6. We examined the possibility that heptanol revealed QEJPs by disrupting electrical coupling between cells in the smooth muscle syncytium. Heptanol (2.0 mM) had no effect on the amplitude distribution, time courses, or the frequency of occurrence of SEJPs. Intracellular input impedance (Rin) of smooth muscle cells was left unaltered by heptanol. 7. 'Cable' potentials of the vas deferens, recorded using the partition stimulation method, also remained unchanged in the presence of heptanol. Thus, heptanol appeared not to modify syncytial electrical properties of the smooth muscle in any significant way. 8. Our observations show directly that the quantal depolarizations underlying the EJP in syncytial smooth muscle are SEJP-like events. However, no unequivocal statement can be made about the mechanism by which heptanol unmasks QEJPs from EJPs. (+info)
Intercellular communication in spinal cord astrocytes: fine tuning between gap junctions and P2 nucleotide receptors in calcium wave propagation.
Electrophysiological properties of gap junction channels and mechanisms involved in the propagation of intercellular calcium waves were studied in cultured spinal cord astrocytes from sibling wild-type (WT) and connexin43 (Cx43) knock-out (KO) mice. Comparison of the strength of coupling between pairs of WT and Cx43 KO spinal cord astrocytes indicates that two-thirds of total coupling is attributable to channels formed by Cx43, with other connexins contributing the remaining one-third of junctional conductance. Although such a difference in junctional conductance was expected to result in the reduced diffusion of signaling molecules through the Cx43 KO spinal cord syncytium, intercellular calcium waves were found to propagate with the same velocity and amplitude and to the same number of cells as between WT astrocytes. Measurements of calcium wave propagation in the presence of purinoceptor blockers indicate that calcium waves in Cx43 KO spinal cord astrocytes are mediated primarily by extracellular diffusion of ATP; measurements of responses to purinoceptor agonists revealed that the functional P2Y receptor subtype is shifted in the Cx43 KO astrocytes, with a markedly potentiated response to ATP and UTP. Thus, the reduction in gap junctional communication in Cx43 KO astrocytes leads to an increase in autocrine communication, which is a consequence of a functional switch in the P2Y nucleotide receptor subtype. Intercellular communication via calcium waves therefore is sustained in Cx43 null mice by a finely tuned interaction between gap junction-dependent and independent mechanisms. (+info)
Roles of gap junctional communication of cumulus cells in cytoplasmic maturation of porcine oocytes cultured in vitro.
Cumulus cells of the oocyte play important roles in in vitro maturation and subsequent development. One of the routes by which the factors are transmitted from cumulus cells to the oocyte is gap junctional communication (GJC). The function of cumulus cells in in vitro maturation of porcine oocytes was investigated by using a gap junction inhibitor, heptanol. Cumulus-oocyte complexes (COCs) were collected from the ovaries of slaughtered gilts by aspiration. After selection of COCs with intact cumulus cell layers and uniform cytoplasm, they were cultured in a medium with 0, 1, 5, or 10 mM of heptanol for 48 h. After culture in vitro, one group of oocytes was assessed for nuclear maturation and glutathione (GSH) content, and another group was assigned to in vitro fertilization and assessed for the penetrability of oocytes and the degree of progression to male pronuclei (MPN) of penetrated spermatozoa. At the end of in vitro maturation, the oocytes reached metaphase II at a high rate (about 80%) regardless of the presence of heptanol at various concentrations. Cumulus cell expansion and the morphology of oocytes cultured in the medium with heptanol were similar to those of control COCs matured without heptanol. The amount of GSH in cultured oocytes tended to decrease as the concentration of heptanol in the medium was increased. Although there was no difference in the rates of penetrated oocytes cultured in media with different concentrations of heptanol, the proportion of oocytes forming MPN after insemination decreased significantly (P < 0.01) at all concentrations tested. A higher rate of sperm (P < 0.01) failed to degrade their nuclear envelopes after penetration into the oocytes that were treated with heptanol. GJC between the oocyte and cumulus cells might play an important role in regulating the cytoplasmic factor(s) responsible for the removal of sperm nuclear envelopes as well as GSH inflow from cumulus cells. (+info)
Gap junctions in isolated rat aorta: evidence for contractile responses that exhibit a differential dependence on intercellular communication.
Connexin43 (Cx43) is a major gap junction protein present in the Fischer-344 rat aorta. Previous studies have identified conditions under which selective disruption of intercellular communication with heptanol caused a significant, readily reversible and time-dependent diminution in the magnitude of alpha1-adrenergic contractions in isolated rat aorta. These observations have indentified a significant role for gap junctions in modulating vascular smooth muscle tone. The goal of these steady-state studies was to utilize isolated rat aortic rings to further evaluate the contribution of intercellular junctions to contractions elicited by cellular activation in response to several other vascular spasmogens. The effects of heptanol were examined (0.2-2.0 mM) on equivalent submaximal ( approximately 75% of the phenylephrine maximum) aortic contractions elicited by 5-hydroxytryptamine (5-HT; 1-2 microM), prostaglandin F2alpha (PGF2alpha; 1 microM) and endothelin-1 (ET-1; 20 nM). Statistical analysis revealed that 200 microM and 500 microM heptanol diminished the maximal amplitude of the steady-state contractile responses for 5-HT from a control response of 75 +/- 6% (N = 26 rings) to 57 +/- 7% (N = 26 rings) and 34.9 +/- 6% (N = 13 rings), respectively (P<0.05), and for PGF2alpha from a control response of 75 +/- 10% (N = 16 rings) to 52 +/- 8% (N = 19 rings) and 25.9 +/- 6% (N = 18 rings), respectively (P<0.05). In contrast, 200 microM and 500 microM heptanol had no detectable effect on the magnitude of ET-1-induced contractile responses, which were 76 +/- 5. 0% for the control response (N = 38 rings), 59 +/- 6.0% in the presence of 200 microM heptanol (N = 17 rings), and 70 +/- 6.0% in the presence of 500 microM heptanol (N = 23 rings) (P<0.13). Increasing the heptanol concentration to 1 mM was associated with a significant decrease in the magnitude of the steady-state ET-1-induced contractile response to 32 +/- 5% (21 rings; P<0.01); further increasing the heptanol concentration to 2 mM had no additional effect. In rat aorta then, junctional modulation of tissue contractility appears to be agonist-dependent. (+info)
Chloride secretion by bovine ciliary epithelium: a model of aqueous humor formation.
PURPOSE: To study the physiological mechanisms of the Cl transport across the bovine ciliary body-epithelium (CBE). METHODS: Fresh isolated bovine CBE was mounted in an Ussing-type chamber. The effects of ion substitution and transport inhibitors on electrical measurements and Cl transport were investigated. RESULTS: The potential difference (PD) across the preparation was 0.55 +/- 0.04 mV and was consistently negative at the aqueous side. The short-circuit current (SCC) and tissue resistance (Rt) were found to be 8.0 +/- 0.7 microA/cm2 and 72 +/- 3 omega/cm2, respectively. Both the PD and the SCC of the bovine CBE were found to depend primarily on the concentration of the Cl bath and to a lesser extent on the Na or HCO3 concentration. At 30 mM Cl, the polarity of the PD and the direction of the SCC were reversed. Reducing the extracellular Na or Cl concentration abolished the net Cl transport into the eye under the short-circuited condition. Bilateral bumetanide (0.1 mM), but not 4,4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS; 0.1 mM), greatly inhibited the Cl transport. Bumetanide, when applied to either side, inhibited the Cl transport. The effect, however, was three times greater on the stromal side than on the aqueous surface. Bilateral heptanol (3.5 mM) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 0.1 mM) on the aqueous side also inhibited the Cl transport by 80% and 92%, respectively. CONCLUSIONS: The results suggest that the major Cl influx pathway at the basolateral border in pigmented epithelial cells is through the Na-K-2Cl cotransporter, but not the Cl/HCO3 and Na/H double exchangers. Intercellular gap junctions between the two cell layers and the NPPB-sensitive Cl channels at the basolateral surface in nonpigmented epithelial cells also play a crucial role in regulating the Cl movement across the functional syncytium. (+info)
Subthreshold oscillation of the membrane potential in magnocellular neurones of the rat supraoptic nucleus.
Electrophysiological properties and ionic basis of subthreshold oscillation of the membrane potential were examined in 104 magnocellular neurones of the rat supraoptic nucleus using intracellular recording techniques in a brain slice preparation. Subthreshold oscillation of the membrane potential occurring in all neurones examined was voltage dependent. Oscillation was initiated 7-12 mV negative to the threshold of fast action potentials. Oscillation was the result of neither excitatory nor inhibitory synaptic activity nor of electric coupling. Frequency analyses revealed a broad band frequency distribution of subthreshold oscillation waves (range 10-70 Hz). The frequency band of 15-33 Hz was observed in neurones depolarized close to the threshold of discharge. Subthreshold oscillation was blocked by TTX (1.25-2.5 microM) as well as by TEA (15 mM). Subthreshold oscillation was not blocked by low Ca(2+)-high Mg(2+) superfusate, CdCl(2), TEA (1-4.5 mM), 4-aminopyridine, apamin, charybdotoxin, iberiotoxin, BaCl(2), carbachol and CsCl. During application of TTX, stronger depolarization induced high-threshold oscillation of the membrane potential at a threshold of about -32 mV. These oscillation waves occurred at a mean frequency of about 35 Hz and were blocked by CdCl(2). Effects of ion channel antagonists suggest that subthreshold oscillation is generated by the interaction of a subthreshold sodium current and a subthreshold potassium current. The generation of high-threshold oscillation during TTX involves a high-threshold calcium current. Subthreshold oscillation of the membrane potential may be important for the inter-neuronal synchronization of discharge and for the amplification of synaptic events. (+info)