Nitric oxide/cGMP-induced inhibition of calcium and chloride currents in retinal pericytes.

In the CNS, contractile pericytes positioned on endothelium-lined lumens appear to play a role in regulating capillary blood flow. This function may be particularly important in the retina where pericytes are more numerous than in other tissues. Despite the importance of pericytes, knowledge of the effects of vasoactive molecules, such as nitric oxide (NO), on the physiology of these cells is limited. Since it is likely that ion channels play a role in the response of pericytes to signaling molecules from other cells, we used the perforated-patch configuration of the patch-clamp technique to record the whole-cell currents of pericytes located on microvessels freshly isolated from the rat retina. We found that voltage-gated calcium currents and calcium-activated chloride currents were inhibited during exposure to the NO donor, sodium nitroprusside (SNP). 8-Bromo-cyclic guanosine monophosphate (cGMP) mimicked these effects. In contrast, neither SNP nor the cGMP analog significantly affected the potassium or nonspecific cation conductances, which establish the resting membrane potential of retinal pericytes. Consistent with endogenous NO suppressing pericyte channel activity, exposure of isolated microvessels to an inhibitor of NO synthase increased the calcium and chloride currents. Since our experiments indicate that chloride channel activity is dependent, in part, upon the function of voltage-gated calcium channels, we postulate that a NO/cGMP-mediated inhibition of calcium channels reduces calcium influx and, thereby, lessens the opening of the calcium-activated chloride channels. This may be one mechanism by which NO decreases the contractile tone of pericytes.

Diabetes-induced disruption of gap junction pathways within the retinal microvasculature.

PURPOSE: Microvascular damage caused by diabetes is a leading cause of visual loss. Identifying events early in the course of diabetic retinopathy may help in understanding and, perhaps, preventing this disorder. The hypothesis that cell-to-cell communication within the retinal microvasculature may be affected soon after the onset of diabetes was tested. METHODS: Streptozotocin was used to induce diabetes in rats. To assess cell-to-cell coupling the gap junction-permeant tracer, Neurobiotin, was delivered via patch pipettes into pericytes located on microvessels freshly isolated from the retinas of diabetic and control animals. Subsequently, immunohistochemical methods revealed the extent of the intercellular spread of the tracer. Electrophysiological methods were also used to detect intercellular communication. RESULTS: In retinal microvessels of control rats, Neurobiotin spread hundreds of micrometers from the tracer-loaded pericytes. However, within days after the onset of diabetes, this cell-to-cell coupling was dramatically reduced. In contrast, microvessels of insulin-treated diabetic rats showed no significant loss of intercellular communication. Consistent with protein kinase C (PKC) playing a role in the diabetes-induced inhibition of gap junction pathways, exposure of microvessels to a PKC activator (phorbol myristate acetate) markedly reduced tracer coupling. CONCLUSIONS: Within retinal microvessels there is extensive cell-to-cell coupling, which is markedly reduced soon after the onset of streptozotocin-induced diabetes. The closure of gap junction pathways disrupts the multicellular organization of retinal microvessels and may contribute to vascular dysfunction.

PDGF-induced coupling of function with metabolism in microvascular pericytes of the retina.

PURPOSE: The aim of this study was to test the hypothesis that platelet-derived growth factor (PDGF)-BB regulates the physiology of retinal pericytes, which are contractile cells located on the abluminal surface of capillaries. The expression of PDGF-BB and its cognate receptor in retinal vessels suggests a vasoactive function. However, although endothelium-derived PDGF-BB appears vital for the development of pericyte-containing microvessels, its role in the mature vasculature remains uncertain. METHODS: Based on the premise that ion channels mediate the responses of pericytes to vasoactive signals, the perforated-patch configuration of the patch-clamp technique was used to determine the effect of PDGF-BB on the ionic currents and membrane potential of pericytes located on microvessels freshly isolated from the adult rat retina. Changes in pericyte calcium levels were monitored with the calcium indicator fluo-4. Differential interference contrast optics and image analysis software aided in assessing the effects of PDGF-BB on the lumens of isolated pericyte-containing microvessels. In some experiments, blockers of adenosine triphosphate (ATP) synthesis created chemical ischemia. RESULTS: Electrophysiological recordings from pericytes showed that PDGF-BB can activate nonspecific cation channels, chloride channels, and ATP-sensitive potassium channels. The metabolic status of an isolated capillary determined which of these ion channels were activated by PDGF-BB and thereby whether the membrane potential decreased or increased, the cell calcium rose or fell, and the vessel lumen constricted or dilated. CONCLUSIONS: The ability of PDGF-BB to be a vasoconstrictor when energy supplies are ample and to be a vasodilator under ischemic conditions may provide an efficient mechanism to link capillary function to local metabolic needs.

Adenosine activates ATP-sensitive K(+) currents in pericytes of rat retinal microvessels: role of A1 and A2a receptors.

In the CNS, contractile pericytes are positioned on the endothelial walls of microvessels where they are thought to play a role in adjusting blood flow to meet local metabolic needs. This function may be particularly important in the retina where pericytes are more numerous than at any other site. Despite the putative importance of pericytes, knowledge of the mechanisms by which vasoactive molecules, such as adenosine, regulate their function is limited. Using the perforated-patch configuration of the patch-clamp technique to monitor the whole-cell currents of pericytes located on microvessels freshly isolated from the adult rat retina, we found that adenosine reversibly activated a hyperpolarizing current in 98% of the sampled pericytes. This adenosine-induced current is likely to be due to the opening of ATP-sensitive potassium (K(ATP)) channels since it had a reversal potential near the equilibrium potential for K(+), was inhibited by the K(ATP) channel blocker, glibenclamide, and was mimicked by pinacidil, which is a K(ATP) channel opener. Experiments with specific agonists and antagonists indicated that both the high affinity A1 and the lower affinity A2a adenosine receptors provided effective pathways for activating K(ATP) currents in pericytes recorded under normal metabolic conditions. However, during chemical ischemia, the A1 receptor pathway rapidly became ineffective. In contrast, activation of A2a adenosine receptors continued to open K(ATP) channels in ischemic pericytes. These results suggest that the regulation of K(ATP) channels via A1 and A2a receptors allows adenosine to serve over a broad range of metabolic conditions as a vasoactive signal in the retinal microvasculature.

Dopamine activates ATP-sensitive K+ currents in rat retinal pericytes.

The relatively sparse vasculature of the retina minimizes obstruction to incoming light, but also poses a challenge to fulfilling the metabolic demands of retinal neurons. An efficient process for distributing energy supplies to areas of need is likely to involve neuron-derived vasoactive signals. However, knowledge of the mechanisms by which capillary perfusion is regulated by neuron-to-vascular signaling is limited. Potential targets of vasoactive molecules released from nerve cells are the pericytes, which are positioned on the endothelial walls of microvessels and are thought to play a role in controlling the microcirculation. In this study, we assessed the effect of dopamine on pericyte physiology. Because dopaminergic neurites are closely associated with microvessels that express dopamine receptors, this molecule is a putative neuron-to-capillary signal, as well as neurotransmitter. We used the perforated-patch configuration of the patch-clamp technique to monitor the whole-cell currents of pericytes located on microvessels freshly isolated from the adult rat retina. In 43% (58/134) of the sampled pericytes, we found that dopamine reversibly activated a hyperpolarizing current, which increased the membrane potential by 19 +/- 1 mV. This dopamine-induced current was inhibited by the ATP-sensitive potassium (KATP) channel blocker, glibenclamide. Consistent with a signaling pathway involving D1 dopamine receptors, adenylate cyclase and protein kinase A (PKA), the selective D1 antagonist, SCH23390, inhibited the hyperpolarizing effect of dopamine; the activator of adenylate cyclase, forskolin, mimicked the dopaminergic effect, and H89, which inhibits PKA, significantly reduced the hyperpolarization induced by dopamine. Taken together, our experiments indicate that a mechanism involving D1 dopamine receptors, adenylate cyclase, and PKA activates KATP currents in retinal pericytes. Our observations support the hypothesis that, in addition to being a neuromodulator, dopamine also serves as a signal linking neuronal activity with the function of the pericyte-containing microvasculature.

Platelet-derived growth factor-BB: a survival factor for the retinal microvasculature during periods of metabolic compromise.

PURPOSE: The aim of this study was to test the hypothesis that platelet-derived growth factor (PDGF) reduces ischemia-induced damage to cells in the retinal microvasculature. METHODS: As a model of ischemia, pericyte-containing microvessels freshly isolated from the adult rat retina were exposed to the inhibitors of ATP synthesis, iodoacetate and antimycin A. Cell viability was assayed by trypan blue exclusion. RESULTS: PDGF-BB significantly reduced cell death induced by chemical ischemia. The half-maximally effective concentration was approximately 15 pM. In contrast to PDGF-BB, which is the specific ligand for PDGF-beta receptors, ischemic death was not reduced by PDGF-AA, which does not activate the beta-receptors. The protective effect of PDGF-BB was blocked by tolbutamide, which is an inhibitor of ATP-sensitive potassium (K(ATP)) channels and mimicked by the K(ATP) channel opener, pinacidil. Nifedipine, which blocks voltage-gated calcium channels (VGCC's), also mimicked the protective effect of PDGF-BB. Consistent with PDGF-BB and nifedipine preventing cell death via a common mechanism, i.e., reducing VGCC activity, the maximal effects of this growth factor and the calcium channel blocker were not additive. CONCLUSIONS: Our results indicate that PDGF-BB significantly reduces the vulnerability of retinal microvessels to damage caused by profound ischemia. During episodes of metabolic compromise, it appears likely that the opening of K(ATP) channels via activation of PDGF-beta receptors initiates an adaptive mechanism to enhance the survival of the retinal microvasculature.

Energy metabolism in human retinal Müller cells.

PURPOSE: To measure selected parameters of energy metabolism and adenosine triphosphate (ATP) production in passaged monolayer cultures of human retinal glial (Müller) cells to assess the effects of varying substrate and oxygen availability on the biochemistry and histologic integrity of these cells. METHODS: Confluent Müller cell cultures were incubated for up to 4 hours at 37 degrees C in a modified minimal essential medium (no serum) under aerobic or mitochondrial-inhibited conditions in the presence and absence of 5 mM glucose or in the presence of lactate, pyruvate, glutamate, or glutamine. Cellular ATP levels, lactic acid production, and (14)CO(2) production from labeled glucose or glutamate were measured along with an examination of cellular morphology. Immunohistochemistry with antibodies to glial cell-specific proteins was also performed. Cells were positive for vimentin, but negative for glial fibrillary acidic protein and glutamine synthetase. RESULTS: Human Müller cells maintained ATP content aerobically at the same level for 4 hours in the presence and absence of glucose. ATP content was also maintained anaerobically at a value equal to that found aerobically, but only in the presence of glucose. ATP content in human Müller cells declined to a very low level when glycolysis was blocked by iodoacetate, and inclusion of lactate, pyruvate, glutamate, or glutamine did not restore the level of ATP. Aerobically, lactic acid production accounted for 99% of the total glucose used, whereas the oxidation of glucose by the mitochondria accounted for only 1%. When mitochondria were inhibited with antimycin A, there was only a modest (1.3-fold) increase in the rate of lactic acid production. No significant differences were found in the histologic appearance of the cells after mitochondrial blockade, but there was massive death of cells after inhibition of glycolysis with iodoacetate. CONCLUSIONS: These results suggest that, in the presence of glucose and oxygen, cultured Müller cells obtain their ATP principally from glycolysis and have a low rate of oxygen consumption. This metabolic pattern may spare oxygen for retinal neurons, particularly in the inner nuclear and ganglion cell layers under normal physiological conditions. Furthermore, retinal Müller cells in culture are resistant to anoxia or absence of glucose, which provides a basis for understanding why Müller cells are less susceptible than neurons to ischemia or hypoglycemia.

Physiology of rat retinal pericytes: modulation of ion channel activity by serum-derived molecules.

1. Pericytes, which are contractile cells located on the outer wall of microvessels, are thought to be particularly important in the retina where the ratio of these cells to vascular endothelial cells is the highest of any tissue. Retinal pericytes are of interest since they may regulate capillary blood flow and because their selective loss is an early event in diabetic retinopathy, which is a common sight-threatening disorder associated with dysfunction of the blood-retinal barrier. 2. Although a breakdown in the vascular endothelial barrier is a frequent pathophysiological event, knowledge of the effects of blood-derived molecules on pericyte function is limited. Based on the premise that ion channels play a vital role in cellular function, we examined the effect of serum on the ionic currents of retinal pericytes. To do this, we used the perforated-patch configuration of the patch-clamp technique to monitor the whole-cell currents of pericytes located on freshly isolated rat retinal microvessels. 3. Exposure to serum reversibly activated inward and outward currents in virtually all of the sampled retinal pericytes. Two types of sustained conductances were induced by serum. These were a calcium-permeable non-specific cation (NSC) current and a voltage-dependent potassium current. In addition, exposure to serum increased the activity of chloride channels which caused transient depolarizing currents. 4. Associated with the activation of these conductances, the membrane potential showed a sustained decrease of 10 +/- 2 mV from -56 mV to -46 mV and, also, transient depolarizations to near -30 mV. The serum-induced depolarizations can activate the voltage-gated calcium channels expressed by the retinal pericytes. 5. Calcium-permeable NSC channels appear to play a critical role in the response of pericytes to serum-derived molecules. Consistent with this, activation of the chloride and potassium channels was sensitive to SK&F 96365, which is a blocker of NSC channels. In addition, chloride and potassium channel activation was dependent on extracellular calcium. 6. The effects of serum on the activity of channels in retinal pericytes were qualitatively mimicked by insulin-like growth factor-1 (IGF-1), which is a normal constituent of the blood. 7. There are significant differences in the effects of serum on retinal pericytes compared with vascular smooth muscle cells. Serum activated sustained conductances in retinal pericytes but not in the vascular smooth muscle cells. This suggests a fundamental difference in the mechanisms by which serum-derived molecules affect these two types of cells. 8. We conclude that serum-derived molecules, such as IGF-1, can activate several types of ion channels in retinal pericytes. These changes in channel activity are likely to influence pericyte function at sites of a breakdown in the blood-retinal barrier.

Plasma-induced changes in the physiology of mammalian retinal glial cells: role of glutamate.

Plasma can leak into the nervous system when the vascular endothelial barrier is compromised. Although this occurs commonly, little is known about the effects of plasma on the function of cells in the central nervous system. In this study, we focused on the responses of glial cells, which, because they ensheathe the blood vessels, are the first cells exposed to leaking plasma. We used the perforated-patch configuration of the patch-clamp technique to assess the effects of plasma on freshly dissociated bovine and human Müller cells, the principal glia of the retina. To monitor the function of Müller cells in situ, we recorded electroretinograms from isolated retinas. We found that plasma activates an electrogenic glutamate transporter and inhibits inward-rectifying K+ channels, as well as a transient outward current. Glutamate, a normal constituent of the blood, mimicked these effects. Unlike our recent findings with serum, which contains molecules generated by the clotting process, plasma neither activated a nonspecific cation conductance nor inhibited the slow P(III) component of the electroretinogram, which is generated by Müller cells responding to light-evoked changes in the extracellular potassium concentration ([K+]o). Taken together, our observations indicate that a leakage of serum into the retina compromises the regulation of [K+]o by Müller cells; however, when plasma enters the retina at sites of a breakdown in the blood-retinal barrier, these glia can maintain K+ homeostasis while reducing the potentially neurotoxic levels of glutamate.

Expression of transforming growth factor-beta s and their receptors by human retinal glial cells.

PURPOSE: To help test the hypothesis that transforming growth factor beta (TGF-beta) may serve an autocrine function in the retina, we asked whether human Müller (glial) cells in culture express TGF-beta receptors, contain transcripts for various isoforms of this cytokine, and release TGF-beta s into the medium. METHODS: Using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique with specific primers for TGF-beta 1, -beta 2 and -beta 3 precursors and for TGF-beta type I and type II receptors, we searched for mRNA transcripts expressed by cultured human Müller cells. Also, an ELISA assay allowed quantification of the levels of various TGF-beta s in medium exposed to these glial cells. RESULTS: Human Müller cells in culture express transcripts for both type I and type II TGF-beta receptors and also for TGF-beta 1 and TGF-beta 2. In conditioned medium, the concentration of TGF-beta 1 in the mature form was below detectable levels, and the total TGF-beta 1 was relatively low (mean = 252 pg/ml in confluent cultures). In contrast, the mean levels of mature (55 pg/ml) and total (2530 pg/ml) TGF-beta 2 were markedly higher. CONCLUSIONS: Our observations that cultured Müller cells contain mRNA coding for the TGF-beta 2 precursor, release TGF-beta 2 into the medium and express transcripts for both type I and type II TGF-beta receptors are consistent with the idea that this cytokine serves an autocrine function for these glia in the retina.

Serum-induced changes in the physiology of mammalian retinal glial cells: role of lysophosphatidic acid.

1. With a breakdown of the vascular-CNS barrier, serum enters the nervous system. Although this is a frequent pathophysiological event, knowledge of the effects of serum on the function of the nervous system is limited. In this study, we examined the effects of serum on the activity of ion channels in Müller cells: the principal glia of the retina. 2. Freshly dissociated Müller cells from the bovine and human retina were studied with the perforated-patch configuration of the patch clamp technique. In other experiments, electroretinograms (ERGs) were recorded from isolated rat retinas. 3. Perforated-patch recordings revealed that serum induced a calcium-permeable, non-specific cation (NSC) current. Approximately 40 s after induction of this current, an outwardly rectifying K+ current was also detected. Sensitivity to charybdotoxin and margatoxin indicated that this K+ current was due to the activation of Kv1.3 channels. This increase in the Kv1.3 current was dependent on extracellular calcium. 4. The NSC and Kv1.3 currents were activated by serum in 100% and 95% of the sampled Müller cells, respectively. Also, in a minority (21%) of the cells, the inwardly rectifying K+ current was inhibited slightly. These changes in ion channel activity were associated with depolarization of the Müller cells. 5. We hypothesized that activation of NSC channels would reduce the siphoning of K+ via the Müller cells. Consistent with this idea, ERGs from isolated retinas showed serum-induced reductions in the slow PIII component, which is generated by Müller cells responding to light-evoked changes in the extracellular K+ concentration. 6. Lysophosphatidic acid (LPA), a component of serum, had effects on Müller cells that were qualitatively similar to those induced by serum. 7. Our observations demonstrate that exposure to serum alters the activity of multiple types of ion channels in Müller glial cells of the mammalian retina. When there is a breakdown of the blood-retina barrier, LPA may be one of the serum-derived molecules which regulates the physiology of Müller cells.

Intracellular ATP activates inwardly rectifying K+ channels in human and monkey retinal Müller (glial) cells.

1. In the vertebrate retina, the inwardly rectifying K+ (KIR) channels of the Müller (glial) cells are pathways for the redistribution of excess extracellular K+. Due to this role in K+ homeostasis, the activity of Müller cell KIR channels is likely to have significant functional consequences for the retina. In this study we asked whether intracellular ATP regulates the function of KIR channels expressed by Müller cells, the principal glia of the retina. 2. Freshly dissociated Müller cells from the human and monkey (Macaca fascicularis) retina were studied with various configurations of the patch-clamp technique. 3. Whole-cell recordings from Müller cells revealed that a run-down of the inwardly rectifying K+ current (IK(IR)) was prevented if the pipette solution contained Mg-ATP. Chemical ischaemia induced by inhibitors of glycolysis and oxidative phosphorylation caused a nearly 10-fold reduction in the IK(IR)) that was fully restored when metabolically inhibited Müller cells were internally perfused with ATP. 4. In recordings from membrane patches of fresh primate Müller cells, we found that inward-rectifying channels with a conductance of 20 pS in 100 mM Ko+ were the predominant type of KIR channel. In excised patches these 20 pS KIR channels were activated when Mg-ATP was at the cytoplasmic surface. Experiments with inside-out patches indicated that the activity of the 20 pS KIR channels can be maintained by ATP synthesized at sites located close to the channel. 5. The inability of the non-hydrolysable ATP analogue 5'-adenylylimidodiphosphate (AMP-PNP) to prevent the run-down of IK(IR))and the Mg2+ dependence of the ATP effect on KIR channels are consistent with a mechanism of activation requiring the hydrolysis of ATP. 6. These observations suggest that the metabolic state of a Müller cell regulates the activity of its 20 pS KIR channels and thus influences the function of the glial cell in maintaining K+ homeostasis in the retina.

cGMP-mediated effects on the physiology of bovine and human retinal Müller (glial) cells.

1. Whole-cell currents of freshly dissociated or cultured Müller cells from human and bovine retinas were studied using the perforated-patch and standard whole-cell recording techniques. 2. We found that internal perfusion of cGMP or external exposure to 8-bromo-cGMP activated a calcium permeable, non-selective cation current in Müller cells, the principal glial cells of the retina. In addition, the activity of calcium-activated potassium channels increased markedly. These currents were minimally affected by cAMP. 3. Molecular studies using the reverse transcription-polymerase chain reaction demonstrated that human müller cells in culture contain transcripts closely related to the rod cyclic nucleotide-gated (CNG) channel. 4. Since guanylate cyclase is a known target for nitric oxide (NO), we tested the effect of NO donors on Müller cell currents. These agents induced currents that were qualitatively similar to those activated by cGMP. 5. Our experiments support the idea that the NO-cGMP pathway regulates the physiology of Müller cells and may play a role in integrating neuron-glia interactions in the retina.

Replication of human cytomegalovirus in human retinal glial cells.

PURPOSE: The purpose of these studies was to characterize the replication cycle of human cytomegalovirus (HCMV) in human retinal glial cells in vitro. METHODS: Cultured human retinal glial cells were exposed to HCMV strain AD169 or low-passage clinical isolates for a 2-hour adsorption period and then incubated in the appropriate growth medium at 37 degrees C. Cultures were examined by microscopy for cytopathic effect and by immunofluorescence staining using monoclonal antibodies directed against immediate-early, early, and late HCMV proteins. Viral DNA was analyzed by field inversion gel electrophoresis and detected using Southern blot analysis or the polymerase chain reaction. RESULTS: Immunocytochemical staining revealed that the glial cells expressed all three classes of HCMV proteins and that infectious virus could be transferred from the medium of the infected cultures to susceptible MRC-5 cell monolayers. Less than 1% of the glial cells expressed the S-phase enzyme, thymidine kinase, at the time of infection compared to MRC-5 fibroblasts, of which 81% expressed it. Progeny virus was found to be highly cell associated in glial cells (80%) at peak virus titer compared to MRC-5 cells (39% cell associated at peak titer). Four low-passage clinical isolates of HCMV from patients with acquired immune deficiency virus also productively infected cultures of human retinal glial cells. Field inversion gel electrophoresis of HCMV-infected glial cell lysates was performed to identify the replicative forms of DNA. Southern blots probed with HCMV-specific probes showed that HCMV DNA replication proceeds through high molecular weight intermediates before forming the 230-kb unit length genome. CONCLUSIONS: The full permissive replication of HCMV in human retinal glial cells indicates that glial cells are a likely site of HCMV replication in the retina and thus may play an important role in the pathogenesis of HCMV retinitis.

Maintenance of replicative intermediates in ganciclovir-treated human cytomegalovirus-infected retinal glia.

OBJECTIVES: To characterize the molecular structure of the human cytomegalovirus (HCMV) DNA maintained in cultures of human retinal glia following ganciclovir treatment and to determine the biological activity of the DNA. METHODS: Cultures of human retinal glia were established, infected with HCMV, treated with ganciclovir, and embedded in agarose, and the viral DNA was analyzed by field inversion gel electrophoresis. RESULTS: The HCMV DNA was found to persist in cultures of infected, ganciclovir-treated retinal glial cells in the form of replicative intermediates. After removal of ganciclovir, processed forms of DNA in the 500-to 1000-kilobase range were found as well as 230-kb unit length genome. Infectious virus was recovered after termination of ganciclovir treatment. CONCLUSION: The data are consistent with the concept that ganciclovir's virostatic nature permits maintenance of HCMV DNA in retinal glia in a biologically active form that is capable of replication after removal of the drug.

Characterization of an L-type calcium channel expressed by human retinal Müller (glial) cells.

The traditional notion that glial cells are permeable only to potassium has been revised. For example, glia from various parts of the nervous system have calcium-permeable ion channels. Since characterization of the calcium channels in glia is limited, the purpose of this study was to determine the molecular identity and examine the functional properties of a voltage-gated calcium channel expressed by Müller cells, the predominant glia of the retina. Whole-cell and perforated-patch recordings of human Müller cells in culture revealed a high threshold voltage-activated calcium current that is blocked by dihydropyridines, but not by omega-conotoxin GVIA or omega-conotoxin MVIIC. RT-PCR of cultured human Müller cells using primers specific for the calcium channel subunits demonstrated the expression of an L-type channel composed of the alpha 1D, alpha 2 and beta 3 subunits. The alpha 2 subunit of the Müller cell calcium channel is a splice variant which is distinct from either the skeletal muscle alpha 2s or the brain alpha 2b. Our electrophysiological experiments indicate that the alpha 1D/alpha 2/beta 3 calcium channel is functionally linked with the activation of a potassium channel that may serve as one of the pathways for the redistribution by Müller cells of excess retinal potassium.

Activation of NMDA receptor-channels in human retinal Müller glial cells inhibits inward-rectifying potassium currents.

Although it is well known that neurotransmitters mediate neuron-to-neuron communication, it is becoming clear that neurotransmitters also affect glial cells. However, knowledge of neuron-to-glial signalling is limited. In this study, we examined the effects of the glutamate agonist N-methyl-D-aspartate (NMDA) on Müller cells, the predominant glia of the retina. Our immunocytochemical studies and immunodetection by Western blotting with monoclonal antibodies specific for the NMDAR1 subunit provided evidence for the expression by human Müller cells of this essential component of NMDA receptor-channels. Under conditions in which potassium currents were blocked, NMDA-induced currents could be detected in perforated-patch recordings from cultured and freshly dissociated human Müller cells. These currents were inhibited by competitive and non-competitive blockers of NMDA receptor-channels. Extracellular magnesium reduced the NMDA-activated currents in a voltage-dependent manner. However, despite a partial block by magnesium, Müller cells remained responsive to NMDA at the resting membrane potential. Under assay conditions not blocking K+ currents, exposure of Müller cells to NMDA was associated with an MK-801 sensitive inhibition of the inward-rectifying K+ current (IK(IR)), the largest current of these glia. This inhibitory effect of NMDA appears to be mediated by an influx of calcium since the inhibition of IK(IR) was significantly reduced when calcium was removed from the bathing solution or when the Müller cells contained the calcium chelator, BAPTA. Inhibition of the Müller cell KIR channels by the neurotransmitter glutamate is likely to have significant functional consequences for the retina since these ion channels are involved in K+ homeostasis, which in turn influences neuronal excitability.

Truncation of IGF-I yields two mitogens for retinal Müller glial cells.

In the CNS only a truncated form of insulin-like growth factor-I (IGF-I) is detected. Although truncated IGF-I (t-IGF-I) retains mitogenicity, growth promoting activities have not been detected for the tripeptide that is cleaved from IGF-I during truncation. Here, we asked whether the tripeptide is itself a growth factor. Using cultured Müller glial cells from the adult human retina, we found that the cleaved tripeptide, glycine-proline-glutamate, stimulated the proliferation of these cells. Pharmacological experiments indicated that this proliferative effect involves activation of N-methyl-D-aspartate (NMDA) receptors. In addition, t-IGF-I was also mitogenic in our culture system and had an EC50 markedly less than that for IGF-I. Thus, truncation of IGF-I may be a mechanism to augment the mitogenic effect of this gene product by creating a more potent variant and a cleaved tripeptide that is itself a mitogen.

Thrombin-induced inhibition of potassium currents in human retinal glial (Müller) cells.

1. Glial cells are known to play a role in regulating the microenvironment of the nervous system. While earlier considerations of glial function assumed a passive, static physiology for these cells, this is not likely to be the case. In this study, we begin to examine how the physiology of Müller glial cells changes in response to molecules in the microenvironment. 2. Perforated-path recordings and intracellular calcium measurements were performed on human retinal Müller cells in vitro. 3. Analysis of whole-cell currents revealed that the human Müller glial cells have an inwardly rectifying K+ current (IK(IR) which is active near the resting membrane potential. This IK(IR) is significantly inhibited when the Müller cell is exposed to thrombin, a molecule that is likely to enter the retina with a breakdown of the blood-retinal barrier and may be endogenous to the nervous system. 4. A variety of experiments point to a role for Ca2+ as a second messenger mediating the inhibitory effect of thrombin on the IK(IR) of Müller cells. Specifically, thrombin evokes an increase in intracellular [Ca2+] in the Müller cells; the Ca2+ chelator BAPTA blocks the effects of thrombin on both the inhibition of IK(IR) and the rise in intracellular [Ca2+]; exposure to ionomycin, a calcium ionophore, induces a reduction in the IK(IR) of Müller cells. 5. A thrombin- induced inhibition in the IK(IR) of Müller cells is likely to have significant functional consequences for the retina since these ion channels are involved in K+ homeostasis. 6. Our experiments support the idea that the physiology of Müller glial cells is dynamic and can be markedly affected by molecules in the microenvironment.

Regulation of retinal glial cell proliferation by antiproliferative molecules.

Glial cells normally do not proliferate in the adult retina despite the presence of glial mitogens. In this study, we examined the hypothesis that endogenous antiproliferative molecules inhibit the effects of glial mitogens. Using cultures of glial cells obtained from the adult human retina, we found that transforming growth factor beta 2 (TGF beta 2) and a metabotrophic glutamate agonist (t-ACPD) inhibit the mitogenic effects of basic fibroblast growth factor, platelet-derived growth factor, epidermal growth factor and insulin-like growth factor-1. These antiproliferative effects may involve activation of protein kinase C (PKC) since chelerythine, a specific PKC inhibitor, blocks the antiproliferative effects of TGF beta 2 and t-ACPD. Furthermore, exposure of the glia to a phorbol ester mimics the inhibitory effects of TGF beta 2 or t-ACPD. Although TGF beta 2 and t-ACPD markedly inhibit a number of mitogens, they do not alter the mitogenic response of retinal glia to thrombin and glutamate. A common characteristic of the mitogens sensitive to TGF beta 2 or t-ACPD is activation of tyrosine kinase-linked receptors. In contrast, thrombin acts at a G-protein-linked receptor, and glutamate stimulates retinal glial proliferation via activation of an NMDA receptor. It appears that TGF beta 2 and t-ACPD may selectively inhibit retinal glial mitogenesis mediated by activation of tyrosine kinase-linked receptors. Our experiments support the idea that endogenous antiproliferative molecules play a role in preventing glial proliferation in the retina.