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Biomedical subjects

D C Spray

Publications and source records attributed to D C Spray.

At least 109 records · Page 6Linked to original sources
PubMed1993

Gap junctions in the brain: where, what type, how many and why?

Gap junctions represent well-documented means of intercellular communication in various tissues, including the brain, where they function as portals allowing the exchange of electrolytes, second messengers and metabolites between cells. In view of the enormous recent surge of information dealing with the cellular and molecular biology of gap junctions in non-nervous tissue, as well as current interest in the cell biology of glia, this review is intended to provide an overview of the molecular and functional implications of gap-junction-mediated intercellular communication in the nervous system.

Animals↗
PubMed1993

Cytokine-induced programmed death of cultured sympathetic neurons.

Programmed cell death (PCD) of sympathetic neurons is inhibited by nerve growth factor. However, factors that induce PCD of these cells are unknown. Leukemia inhibitory factor (LIF) and ciliary neurotrophic factor, neuropoietic cytokines known to regulate sympathetic neuron gene expression, were examined for effects on survival of cultured sympathetic neurons. Treatment with LIF or ciliary neurotrophic factor caused neuronal death in a dose-dependent fashion. Inhibition of RNA or protein synthesis, or treatment with potassium, all of which prevent PCD after nerve growth factor deprivation, prevented LIF-induced death. The morphologic and ultrastructural characteristics of the neuronal death induced by LIF and by nerve growth factor deprivation were similar. Furthermore, LIF treatment resulted in DNA fragmentation with a characteristic "ladder" on Southern blot analysis. These observations suggest that neuron numbers may be regulated by factors which initiate PCD, as well as by factors which prevent it.

Analysis of Variance↗
PubMed1993

Gap junctions formed of connexin43 are found between smooth muscle cells of human corpus cavernosum.

Despite sparse autonomic innervation, the smooth muscle cells of the corpus cavernosum relax and contract synchronously to achieve penile erection and flaccidity. As with other smooth muscle cell types, the excitation process in the corpora is presumably propagated through gap junctions to allow the diffusion of current-carrying ions and second messenger molecules from cell to cell. Using both molecular and immunocytochemical techniques, we have identified gap junctions between human corporal smooth muscle cells in situ and in culture. Northern analyses demonstrated that corporal smooth muscle cells express the gap junction protein connexin43, but not connexin26 mRNA. Immunoblots showed the presence of connexin43 isoforms, whereas connexin32 was not detected. Immunocytochemical studies in cultured cells identified prominent connexin43 immunoreactive puncta between cells, as well as within the cytoplasm. In addition, gap junction membranes both in situ and in culture were labelled in thin section by anti-connexin43 antibodies using the immunogold technique. We conclude that the presence and distribution of gap junctions in this sparsely innervated tissue may provide an important mechanism of intercellular communication among the smooth muscle cells, and thus play a major role in coordinating tissue contraction and relaxation.

Blotting, Northern↗
PubMed1993

Gating characteristics of a steeply voltage-dependent gap junction channel in rat Schwann cells.

The gating properties of macroscopic and microscopic gap junctional currents were compared by applying the dual whole cell patch clamp technique to pairs of neonatal rat Schwann cells. In response to transjunctional voltage pulses (Vj), macroscopic gap junctional currents decayed exponentially with time constants ranging from < 1 to < 10 s before reaching steady-state levels. The relationship between normalized steady-state junctional conductance (Gss) and (Vj) was well described by a Boltzmann relationship with e-fold decay per 10.4 mV, representing an equivalent gating charge of 2.4. At Vj > 60 mV, Gss was virtually zero, a property that is unique among the gap junctions characterized to date. Determination of opening and closing rate constants for this process indicated that the voltage dependence of macroscopic conductance was governed predominantly by the closing rate constant. In 78% of the experiments, a single population of unitary junctional currents was detected corresponding to an unitary channel conductance of approximately 40 pS. The presence of only a limited number of junctional channels with identical unitary conductances made it possible to analyze their kinetics at the single channel level. Gating at the single channel level was further studied using a stochastic model to determine the open probability (Po) of individual channels in a multiple channel preparation. Po decreased with increasing Vj following a Boltzmann relationship similar to that describing the macroscopic Gss voltage dependence. These results indicate that, for Vj of a single polarity, the gating of the 40 pS gap junction channels expressed by Schwann cells can be described by a first order kinetic model of channel transitions between open and closed states.

Animals↗
PubMed1993

Gap junctions between human corpus cavernosum smooth muscle cells: gating properties and unitary conductance.

We previously showed that corpus cavernosum smooth muscle cells are connected via gap junctions in situ and in culture and that a major protein component of these gap junctions is connexin43. To characterize the physiological properties of the gap junctions between corpus cavernosum smooth muscle cells, we now demonstrate that the cells are dye and electrically coupled and describe some of the gating properties of these gap junctional channels at macroscopic and single-channel levels. Junctional conductance (gj) between corporal smooth muscle cells was moderately voltage sensitive; was reduced rapidly, reversibly, and completely by halothane; and was increased by treatment with a tumor-promoting phorbol ester [12-O-tetradecanoylphorbol-13-acetate (TPA)] and decreased by isoproterenol. Histograms of unitary junctional currents revealed multiple conductance peaks with events of approximately 90 pS being the most abundant. TPA and phenylephrine produced large increases in relative frequencies of the smaller events, whereas isoproterenol and 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP) slightly increased the relative frequencies of the larger events. None of the tested drugs substantially affected the steady-state voltage dependence of gj. These second messenger systems also affected expression of connexin43 by corpus cavernosum smooth muscle cells, as judged by immunoblots. At 6 h of treatment, both TPA- and 8-BrcAMP-treated cultures showed markedly elevated levels of connexin43, whereas at 24 h, the level of connexin43 in TPA-treated cultures had returned to control levels. Together, these data indicate that second messenger molecules involved in penile erection produce changes in gap junction expression and function; it is plausible that these changes could be physiologically relevant in altering and propagating changes in vasomotor tone.

Cell Communication↗
PubMed1993

The role of gap junctions and ion channels in the modulation of electrical and chemical signals in human corpus cavernosum smooth muscle.

Intercellular communication through aqueous intercellular channels, known as gap junctions, has been postulated to provide an important mechanism for coordinating the rapid and synchronous responses of corporal smooth muscle during human penile erection and detumescence. Mathematical modeling analyses of drug diffusion were utilized to examine the potential physiological importance of the intercellular pathway to the regulation of smooth muscle tone in the human corpus cavernosum. In addition, patch clamp analyses and optical imaging studies were conducted to assess the ionic basis for cellular excitability and homeostasis in cultured corporal smooth muscle cells. In short, the computer modeling studies demonstrated that intercellular communication through gap junctions is likely to be the 'preferred' pathway for coordination of cellular activation and syncytial smooth muscle responses in this tissue. Moreover, the observed ion channel diversity reveals even further complexities to the modulation of corporal smooth muscle tone.

Calcium Channels↗
PubMed1993

Gap junctions modulate tissue contractility and alpha 1 adrenergic agonist efficacy in isolated rat aorta.

Immunocytochemical analysis, using antibodies directed against connexin43, revealed abundant gap junctions between smooth muscle cells in intact aorta from Fischer 344 rats. Therefore, the authors evaluated the potential contribution of these intercellular junctions to contractile responses elicited by alpha 1 adrenergic receptor activation in rat aortic rings. Preincubation with the selective junctional uncoupling agent heptanol (200 microM) diminished the magnitude of contractions induced by the low-efficacy partial agonist oxymetazoline (1-3 microM) by 50.6 +/- 4.5% (P < .01; n = 16 rings from 16 rats) but had no effect on equivalent contractions induced by the high-efficacy agonist phenylephrine (0.1 microM; n = 16 rings from 16 animals). Reduced phenylephrine contractility was observed at higher heptanol concentrations (500 microM). However, neither 200 nor 500 microM heptanol altered the magnitude of contractions elicited by 60 mM KCl, indicating that tissue contractility per se was unaffected by heptanol. In calcium-free solution, the magnitude of the phasic contraction induced by phenylephrine was three-fold greater than the magnitude of the oxymetazoline-induced phasic contraction (P < .001) but the phasic responses to both agonists were unaffected by the same heptanol concentrations that significantly diminished their steady-state responses. Because heptanol, at the concentrations used, has selective pharmacological actions on gap junctions, these studies provide additional support for a role of gap junctions in the maintenance and modulation of vasomotor tone. In rat aorta, junctional transfer of alpha 1 adrenergic-receptor activated second-messenger molecules appears to be an important modulator of tissue contractility and agonist efficacy.

Adrenergic alpha-Agonists↗
PubMed1993

Biophysical properties of the human cardiac gap junction channel.

1. Gap junction channels interconnect cells of the pacemaking, conduction and contraction elements of the heart and also endothelial and smooth muscle cells of vasculature, thereby providing pathways for electrotonic current spread and for second messenger diffusion. The major gap junction protein in the cardiovascular system is connexin43. 2. When human connexin43 is stably expressed in pairs of a communication-deficient cell line (SKHep1) channels are produced with unitary conductance (gamma j), lipophile sensitivity and voltage-dependent gating similar to those of mammalian systems in which connexin43 is endogenously expressed. 3. At moderate transjunctional voltages (Vj), two gamma j values dominated the recordings, about 60 and 90 pS with CsCl patch solution. The smaller channel size is favored by phosphorylating treatments and the larger channel, by dephosphorylating treatments. 4. Human connexin43 mutants truncated at the carboxy termini display a change in gamma j while a point mutation in the third transmembrane spanning domain appears to change channel selectivity. 5. Voltage dependence of the human connexin43 channel is marked at Vjs, above +/- 50 mV, but large residual conductance remains (due probably to a voltage-insensitive substate) even at the largest Vj values; kinetic but not steady-state behavior is affected by phosphorylation state.

Animals↗
PubMed1992

Heterogeneity in gap junction expression in astrocytes cultured from different brain regions.

Heterogeneity among astrocytes suggests that their role in the central nervous system is more complex than is commonly recognized. This paper describes just such a functional difference, comparing gap junctions in astrocytes derived from two brain regions. Astrocytes, both in situ and in culture, employ gap junctions as a means of intercellular communication. Recent evidence utilizing cultured rat cortical and striatal astrocytes has shown that these channels consist of subunits of connexin 43, the same protein as that composing cardiac gap junctions. Here we report that astrocytes cultured from neonatal rat hypothalamus contain a greater number of functional channels than astrocytes from the striatum, a difference reflected in both connexin 43 protein and mRNA. Specifically, in hypothalamic astrocytes the level of connexin 43 protein was approximately four times that found in comparable cultures from the striatum, as determined by immunoblotting. Complementary results from immunocytochemical experiments using an antibody specific for connexin 43 reveal significantly greater fluorescence in astrocytes cultured from the hypothalamus as compared to those from the striatum. Northern blot analysis showed that connexin 43 mRNA levels were also approximately 4-fold greater in the hypothalamic cultures, consistent with the difference seen by immunoblotting. Finally, dye coupling studies using confluent cultures consistently showed that within 1 min Lucifer Yellow injected into striatal astrocytes spread to immediately surrounding cells while in hypothalamic astrocytes dye often spread to apparent third or fourth order neighbors within the same time period. Thus, the higher level of connexin 43 expression seen in hypothalamic astrocytes results in cells with greater numbers of functional channels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Gap junctional communication of primary human keratinocytes: characterization by dual voltage clamp and dye transfer.

We have compared dye coupling in pairs of small (less than 10 microns in diameter) and large (greater than 20 microns in diameter) keratinocytes isolated from normal human epidermis, using Lucifer yellow microinjection. Under control conditions, dye coupling was found in only 1 out of the 25 small pairs tested, whereas it was evident in 75% of the large pairs (n = 52). After a 30-min incubation of the latter pairs in the presence of 10(-6) and 10(-4) M all-transretinoic acid (RA), the percentage of coupling was 53% (n = 15; NS) and 7% (n = 14; P less than 0.001), respectively. The almost complete uncoupling observed after 10(-4) M RA was not reversible even 30 min after return to control medium (n = 8). Dual whole-cell patch-clamp recordings from large keratinocyte pairs showed a macroscopic junctional conductance (gj) of 9 +/- 2 nS (n = 43), which was abolished by heptanol (3.5 mM) in a fully reversible way. Compared to heptanol, 10(-4) M RA abolished keratinocyte gj more slowly and irreversibly (n = 10). By contrast, 10(-6) M RA had no significant effect on gj (n = 8). Single-gap junctional channels were also identified between large keratinocytes. Events histograms of 152 transitions from three experiments revealed three main unitary conductances (gamma j) of 45 +/- 4, 78 +/- 4, and 106 +/- 7 pS. The dye coupling results indicate that junctional communication is markedly different in pairs of small and large cells, which showed the phenotype and keratin markers of basal and suprabasal keratinocytes, respectively. In the latter cell type, coupling is ensured by channels of three sizes and is blocked irreversibly by pharmacologic concentrations of RA.

Alcohols↗
PubMed1992

Gap junction-mediated intercellular diffusion of Ca2+ in cultured human corporal smooth muscle cells.

Ratio imaging using the calcium-sensitive probe fura-2 was employed to study intracellular calcium concentrations and intercellular calcium flux through gap junctions in homogeneous vascular smooth muscle cell cultures derived from the human corpora cavernosa. Microinjection techniques demonstrated that fura-2 free acid was freely diffusible through gap junctions between cultured cells. The resting intracellular calcium level in fura-2-loaded cells was 176.9 +/- 10.5. A robust increase in intracellular calcium was seen in response to both phenylephrine and the calcium ionophore A23187. Microinjection of Ca2+ into individual smooth muscle cells always resulted in significant, although temporally delayed, increases in intracellular calcium levels in adjacent cells; this intercellular calcium flux was reversibly blocked by inhibition of gap junctional communication with 2 mM heptanol. However, although microinjection of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] into individual smooth muscle cells always produced significant increases in intracellular calcium levels in the injected cell, the intercellular spread of Ca2+ in response to Ins(1,4,5)P3 was more variable than for Ca2+ injections. These studies demonstrate that Ca2+, and perhaps Ins(1,4,5)P3 as well, can diffuse between smooth muscle cells through gap junction channels.

Alcohols↗
PubMed1992

Gap junction distribution is altered between cardiac myocytes infected with Trypanosoma cruzi.

Conduction disturbances frequently accompany both acute and chronic Chagas' disease. To explore the possibility that changes in gap junction distribution or abundance might play a role in these disturbances, we have investigated intercellular communication between rat neonatal cardiac myocytes in cultures infected with Trypanosoma cruzi. Contractile activity of infected cells was characterized by regional asynchrony within the culture as well as by irregular contraction patterns. Junctional conductance between infected cell pairs was found to be significantly lower than in uninfected cell pairs, and the rapidity and extent of intercellular transfer of the dye lucifer yellow was markedly reduced between infected cells. Immunocytochemical studies demonstrated that the parasitic infection significantly decreased connexin43 expression at junctional membrane regions, correlating with the detected functional uncoupling. These findings of reduced gap junction abundance and function in trypanosome-infected cells may provide important insight into the pathogenesis of the cardiac arrhythmias that attend Chagas' disease.

Animals↗
PubMed1992

Properties of channels from rat liver gap junction membrane fractions incorporated into planar lipid bilayers.

1. Rat liver membrane fractions highly enriched for gap junctions can be incorporated into planar lipid bilayers exhibiting channel currents with both voltage-dependent and independent components. Voltage dependence, however, is only one of the characteristics of liver gap junction channels. Other features include poor ionic selectivity and sensitivity to calcium, pH, octanol and to some intracellularly applied antibodies. 2. To further test the junctional nature of channels from membrane fractions highly enriched in gap junctions incorporated into lipid bilayers we studied the sensitivity of these channels to uncoupling agents and determined channel selectivity properties. 3. We found the incorporated channels to be insensitive to calcium and octanol, and in most cases to pH in the range of 5-7, suggesting that either these agents do not interact directly with the junctional channels or that the corresponding gating regions are inactivated during the isolation and reconstitution procedures. 4. Attempts to block channel activity using polyclonal and monoclonal connexin 32 antibodies were generally unsuccessful, although one antibody (a monoclonal directed against the carboxy terminus portion of connexin32) blocked channel activity. 5. Selectivity measurements indicated that the incorporated channels were slightly cation selective (PNa = PK > PCl) and were permeable to large ions. 6. These results further support the idea that functional connexin32 gap junction channels are present in channel activity recorded from rat liver junctional membranes incorporated into planar bilayers.

Animals↗
PubMed1992

Transcriptional and posttranscriptional control of connexin mRNAs in periportal and pericentral rat hepatocytes.

Distinct patterns of expression of gap junction, or connexin, mRNAs were observed in periportal vs. pericentral hepatocytes. The two cellular fractions (isolated from rat livers by perfusion) were more than 90% parenchymal, as determined by flow cytometry for a hepatocyte-specific marker. The periportal and pericentral fractions were identifiable due to enrichment in enzymatic activities previously shown to be differentially expressed in the respective regions of liver. Northern blot analyses revealed that mRNA encoding connexin 26 was 2.8 times more abundant in the periportal than in the pericentral cells, while connexin 32 mRNA was equally distributed. Messenger RNA from each fraction was radiolabeled in order to compare the relative abundance of the connexin mRNAs in each fraction. The ratio of connexin 26 to connexin 32 mRNA in the portal fraction was about 0.085, and in the central fraction about 0.038. Connexin 26 mRNA was transcribed, however, at a faster rate than connexin 32 mRNA by nuclei isolated from both cellular fractions. Connexin 26 mRNA was transcribed at 3.9 times the rate in nuclei from the periportal than from the pericentral cells. These data suggest that while the zonation of connexin 26 mRNA synthesis in liver appears to be controlled transcriptionally, posttranscriptional regulatory mechanisms determine the relative abundance of the connexin mRNAs.

Animals↗