Monica Lupu-Meiri

NITRIC OXIDE SYNTHASE INHIBITORS PROTECT RAT RETINA AGAINST ISCHEMIC INJURY

Elevation of the ocular pressure in the anterior chamber of the rat eye caused major ischemic damage, manifested as changes in retinal morphology. The two most affected structures were the inner plexiform layer, which decreased in thickness by 90%, and the number of ganglion cells, which decreased by 80%. Pretreatment of the animals with N ω‐nitro‐l‐arginine, a nitric oxide (NOS) inhibitor, almost completely abolished the ischemic damage. Administration of aminoguanidine, a NOS inhibitor selective for the inducible enzyme, partially abolished the ischemic damage. Moreover, administration of the NOS inhibitors 1 h after ischemia, also protected the retina from damage, suggesting that similarly acting drugs could be used clinically to limit ischemic injury in humans. We conclude that NOS, and therefore NO, may be involved in the mechanism of ischemic injury to the retina.

Hemispheric asymmetry of rapid chloride responses to inositol trisphosphate and calcium in Xenopus oocytes

Shallow injection of inositol 1,4,5‐trisphosphate (IP3) near the animal pole of the Xenopus oocyte resulted in a large depolarizing current that decayed rapidly. A similar injection near the vegetal pole produced a much smaller response characterized by a significantly slower rate of decay. Injection of CaCl2 near the animal pole of the oocyte resulted in a large depolarizing current characterized by rapid rise and decay times. Injection near the vegetal pole of the cell produced responses that exhibited similar amplitudes but much longer rise and decay times. The protein kinase C (PK‐C) activator, β‐phorbol 12‐myristate 13‐acetate (PMA), significantly enhanced the rapid responses to IP3 injections at either hemisphere but did not affect the amplitudes of the responses to CaCl2. The PK‐C inhibitor 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine (H‐7) had no effect on the responses to CaCl2. These results imply an asymmetric distribution of calcium stores and chloride channels between the two hemispheres of the oocyte.

Calcium entry in Xenopus oocytes : effects of inositol trisphosphate, thapsigargin and DMSO

Agonist- and inositol 1,4,5-trisphosphate (InsP3)-evoked responses in Xenopus oocytes utilize calcium mobilized from cellular stores as well as from the medium. We studied the effect of the status of Ca stores on InsP3-induced Ca entry. Thapsigargin (TG) caused a net increase of 45Ca2+ efflux from oocytes in a time and dose dependent manner (31 and 54% of total label, at 30 and 60 min, respectively). Incubation with TG (60 min) resulted in a complete loss of the response to InsP3 implying that InsP3-sensitive Ca stores were depleted. Challenge with 1.8 mM Ca2+ resulted in a large depolarizing chloride current (1231 +/- 101 nA) which was not further potentiated by InsP3. This suggested that extensive depletion of cellular Ca stores is sufficient to induce maximal entry of extracellular Ca (Cao). Following the injection of InsP3, a much more limited loss of cellular Ca was sufficient to produce large Ca entry. Dimethyl sulfoxide (DMSO) alone, the vehicle used to dissolve TG, did not cause increase in either efflux of 45Ca2+, nor in the Cao-evoked Cl- current. It did, however, markedly potentiate this current following the injection of InsP3. DMSO moderately inhibited InsP3-induced 45Ca2+ efflux from oocytes. Hence, apparent potentiation of Ca entry can be observed without additional depletion of cellular Ca. We conclude that Ca entry may be induced via either stimulation with InsP3 and limited Ca depletion or depletion of a specific and, possibly small, cellular Ca store alone. The mechanism of DMSO potentiation is unknown, but may be important in view of the universal use of this solvent as vehicle.

Dual regulation by protein kinase C of the muscarinic response in Xenopus oocytes.

Muscarinic stimulation of follicle-enclosed oocytes ofXenopus laevis results in a complex response that involves both depolarizing and hyperpolarizing currents (Dascal and Landau 1980). We studied the involvement of protein kinase C (PK-C1) in the regulation of the acetylcholine-evoked rapid (D1) and of the slow (D2) depolarizing chloride (Cl−) currents. In oocytes maintained at −100 mV [the reversal potential of potassium (K+) ions] under two electrode voltage clamp, the PK-C activator 4-β-phorbol 12-myristate 13-acetate (β-PMA, 0.1 μM) stimulated D1 by 99±17% and inhibited D2 by 67±6%, vs. untreated controls. The inactive isomer (α-PMA) or phorbol alone had no significant effect on the components of the muscarinic response. In order to identify the site of the regulation, we have microinjected the intracellular second messenger of calcium mobilization, inositol 1,4,5-trisphosphate (IP3). β-PMA or the diacylglycerol analog, oleoylacetylglycerol (OAG) stimulated the rapid depolarizing current evoked by IP3 by 220±26% and 394±102%, respectively. α-PMA had little if any effect. The calcium-evoked Cl− current in oocytes pre-treated with the divalent cation ionophore A23187 was, on the other hand, inhibited by β-PMA and OAG (by 82±6% and 54±6%, respectively). α-PMA and phorbol had a limited inhibitory effect. β-PMA, but not α-PMA, also mildly inhibited the IP3-evoked increase in45Ca efflux. The intracellular metabolism of IP3 was not affected by exposure to either β-PMA or OAG. In conclusion, PK-C appears to regulate the acetylcholine-evoked Cl− response in a complex pattern: inhibition of the slow (D2) Cl− current (possibly directly on the Cl− channel) and stimulation of the rapid (D1) Cl− current. Both sites of regulation seem to be distal to IP3 metabolism and to IP3-evoked calcium mobilization. Our results are consistent with the possibility that the complex muscarinic response in Xenopus oocyte is mediated by two populations of Cl− channels.

Extracellular calcium participates in responses to acetylcholine in Xenopus oocytes

We tested the contribution of extracellular calcium (Cao 2+) to membrane electrical responses to acetylcholine (ACh) in native Xenopus oocytes. Removal of Cao caused a decrease in both the rapid (D1) and the slow (D2) chloride currents that comprise the common depolarizing response to ACh in native oocyte. The effect of Cao 2+ removal on the muscarinic response was mimicked by the addition of 1 mM Mn2+, an effective antagonist of calcium influx, though not by antagonists of voltage‐sensitive calcium channels. When oocytes were challenged with ACh in Ca2+‐free medium, subsequent addition of 1.8 mM CaCl2 resulted in a rapid, often transient, depolarizing current. Similarly to the Cao 2+‐dependent component of membrane electrical responses, the Ca2+ ‐evoked current was reversibly abolished by Mn2+, though not by antigonists of voltage‐sensitive calcium channels. Depletion of cellular calcium potentiated the Ca2+‐evoked current, implying negative feedback of calcium channels by calcium. Injection of 10–100 fmol ofinositol 1,4,5‐trisphosphate (IP3) resulted in a two‐component depolarizing current. IP3 injection promoted the appearance of Cao 2+‐evoked current that was significantly potentiated by previous calcium depletion. We suggest that activation of cell‐membrane muscarinic receptors causes opening of apparently voltage‐insensitive and verapamil or diltiazem‐resistant calcium channels. These channels may be activated by IP3 or its metabolites, which increase following the activation of cell membrane receptors coupled to a phospholipase C. The channels may be identical to receptor‐operated channels described in other model systems.

Activation of two different receptors mobilizes calcium from distinct stores in Xenopus oocytes

Acetylcholine (ACh) and thyrotropin-releasing hormone (TRH) utilize inositol 1,4,5-trisphosphate (IP3) as a second messenger and evoke independent depolarizing membrane electrical responses accompanied by characteristic 45Ca efflux profiles in Xenopus laevis oocytes injected with GH3 pituitary cell mRNA. To determine whether this could be accounted for by mobilization of calcium from functionally separate stores, we measured simultaneously 45Ca efflux and membrane electrical responses to ACh and TRH in single oocytes. We found that depletion of ACh-sensitive calcium store did not affect the membrane electrical response to TRH and the TRH-evoked 45Ca efflux. Our data suggest that ACh and TRH mobilize calcium from distinct cellular stores in the oocyte. This is the first demonstration in a single cell of strict subcellular compartmentalization of calcium stores coupled to two different populations of cell membrane receptors that utilize the same second messenger.

Two types of intrinsic muscarinic responses in Xenopus oocytes

Oocytes of 40% of Xenopus laevis frogs respond to acetylcholine (ACh). Oocytes of the majority of responders exhibit the common two-component depolarizing muscarinic response (mean amplitude of the rapid component, 54 nA). Oocytes of approximately 10% of the responders (“variant” donors) exhibit a muscarinic response characterized by a very large transient, rapid current (mean amplitude 1242 nA, reversal potential −33 mV). Responses in oocytes of variant donors exhibit further qualitative differences: pronounced desensitization (absent in oocytes of common donors), characteristic prolonged latency (5.4 vs 0.9 s in oocytes of common donors) and marked inhibition of the response by activators of protein kinase C. Rapid responses in oocytes of variant donors are usually increased by treatment with collagenase, which, in common oocytes, often results in a complete loss of the response that correlates with the loss of muscarinic ligand binding. The number of muscarinic receptors was similar in oocytes of both types of donors (2.2 vs 3.0 fmol/oocyte). Also, the responses of oocytes of variant donors to microinjections of CaCl2 or inositol 1,4,5-trisphosphate were similar to those found in cells of common donors. These findings imply that altered receptor number, calcium stores and/or chloride channel density are not responsible for the variant responses. However, ACh caused an sixteen-fold greater efflux of 45Ca in oocytes of variant donors (35 vs 2.2% of total label in oocytes of common donors). Hence, the characteristics of the variant response may be related to a more efficient coupling between receptor stimulation and the mobilization of cellular calcium.

Inverse agonist abolishes desensitization of a constitutively active mutant of thyrotropin-releasing hormone receptor: role of cellular calcium and protein kinase C

C335Stop is a constitutively active mutant of the TRH receptor (TRH‐R). To investigate the mechanism of the decreased responsiveness of C335Stop TRH‐R, we studied cellular Ca2+ concentrations ([Ca2+]i) in AtT20 cells stably transfected with C335Stop TRH‐R cDNA, or Ca2+‐activated chloride currents in Xenopus laevis oocytes expressing this mutant receptor after injection of cRNA. The competitive TRH‐R binding antagonist, chlorodiazepoxide (CDE), was used as an inverse agonist to study the contribution of constitutive activity to desensitization. Acute treatment with CDE resulted in a rapid (within minutes) decrease in [Ca2+]i and an increase in the response amplitude to TRH with no measurable change in receptor density. Conversely, removal of chronically administered CDE caused a rapid increase in [Ca2+]i and a decrease in TRH response amplitude. CDE abolished heterologous desensitization induced by C335Stop TRH‐R on muscarinic m1‐receptor (m1‐R) co‐expressed in Xenopus oocytes. Chelation of extracellular calcium with EGTA caused a rapid decrease in [Ca2+]i and a concomitant increase in the response to TRH in AtT20 cells expressing C335Stop TRH‐Rs. Chelerythrine, a specific inhibitor of protein kinase C (PKC), reversed the heterologous desensitization of the response to acetylcholine (ACh). The phosphoserine/phosphothreonine phosphatase inhibitor, okadaic acid, abolished the effect of chelerythrine. Down‐regulation of PKC by chronic exposure to phorbol 12‐myristate 13‐acetate (PMA) or acute inhibition with chelerythrine caused a partial resensitization of the response to TRH. Western analysis indicated that the α subtype of protein kinase C was down‐regulated in cells expressing C335Stop TRH‐Rs. Following a 5 min exposure to PMA, the residual αPKC translocated to the particular fraction. We propose that cells expressing the constitutively active mutant TRH‐R rapidly desensitize their response, utilizing a mechanism mediated by an increase in [Ca2+]i and PKC.

Functional characterization of mongoose nicotinic acetylcholine receptor α-subunit: resistance to α-bungarotoxin and high sensitivity to acetylcholine

The mongoose is resistant to snake neurotoxins. The mongoose muscle nicotinic acetylcholine receptor (AChR) α‐subunit contains a number of mutations in the ligand‐binding domain and exhibits poor binding of α‐bungarotoxin (α‐BTX). We characterized the functional properties of a hybrid (α‐mongoose/βγδ‐rat) AChR. Hybrid AChRs, expressed in Xenopus oocytes, respond to acetylcholine with depolarizing current, the mean maximal amplitude of which was greater than that mediated by the rat AChR. The IC50 of α‐BTX to the hybrid AChR was 200‐fold greater than that of the rat, suggesting much lower affinity for the toxin. Hybrid AChRs exhibited an apparent higher rate of desensitization and higher affinity for ACh (EC50 1.3 vs. 23.3 μM for the rat AChR). Hence, changes in the ligand‐binding domain of AChR not only affect the binding properties of the receptor, but also result in marked changes in the characteristics of the current.

Agonist-evoked calcium efflux from a functionally discrete compartment in Xenopus oocytes

Agonist-induced calcium (Ca) mobilization is accompanied by Ca efflux, presumably reflecting the rise in Ca concentration at the cytosolic surface of the cell membrane. We studied the relationship between Ca efflux and intracellular Ca mobilization in Xenopus oocytes. Elevation of cytosolic Ca by a direct injection of 1 nmol 45CaCl2 resulted in a typical Ca-activated chloride current, but not in 45Ca efflux. This demonstrated that a Ca rise at the cytoplasmic surface of the membrane is not sufficient to produce an increased efflux. Co-injection of inositol 1,4,5-trisphosphate (InsP3), to prevent rapid Ca sequestration, also failed to cause Ca efflux. Smaller amounts of labelled Ca (0.05 nmol) equilibrated with Ca stores in a time-dependent pattern with an optimum at 2 h after injection. In contrast, Ca taken up from the medium was immediately available for agonist- or InsP3-induced efflux. Emptying the agonist-sensitive stores with thapsigargin (TG) did not affect chloride currents induced by Ca injection, indicating that these currents were due to direct elevation of Ca at the plasma membrane, rather than Ca-induced Ca release from InsP3-sensitive stores. Agonist-induced depletion of Ca stores enhanced uptake from the extracellular medium and the subsequent release of the label by an agonist. Similar protocol when the label was injected into the oocytes, failed to affect agonist induced efflux. We suggest that, under physiological conditions, agonist-dependent Ca extrusion or uptake in oocytes is executed exclusively via a functionally restricted compartment, which is closely associated with both agonist-sensitive Ca stores and the plasma membrane.