Hagit Shapira

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.

Neuromedin B receptor, expressed in Xenopus laevis oocytes, selectively couples to Gαq and not Gα11

G‐proteins of the q family have been implicated as mediators of bombesin receptors action. We cloned Xenopus Gαq and Gα11 and specifically disrupted the synthesis of either protein with selective antisense oligonucleotides. Gαq antisense inhibited responses mediated by neuromedin B receptor (NMB‐R) by 74%, though not by gastrin‐releasing peptide receptor (GRP‐R). Gα11 antisense had little effect on either GRP‐R‐ or NMB‐R‐mediated responses. This suggests that NMB‐R couples to Gαq, and that GRP‐R and NMB‐R show distinct G‐protein coupling preferences in the Xenopus oocyte.

A human gene encodes a putative G protein-coupled receptor highly expressed in the central nervous system

The mammalian bombesin (Bn)-like neuropeptide receptors gastrin-releasing peptide receptor (GRP-R) and neuromedin B receptor (NMB-R) transduce a variety of physiological signals that regulate secretion, growth, muscle contraction, chemotaxis and neuromodulation. We have used reverse transcription-polymerase chain reaction (PCR) to isolate a cDNA from human brain mRNA, GPCR/CNS, that encodes a putative G protein-coupled receptor (GPCR) based upon the presence of the paradigmatic seven heptahelical transmembrane domains in its predicted amino acid sequence. Analysis of the deduced protein sequence of GPCR/CNS reveals this putative receptor to be 98% identical to the deduced amino acid sequence of a recently reported gene product and minimally identical (approximately 23%) to both murine GRP-R and human endothelin-B (ET-B) receptor. Our deduced protein sequence differs at 12 positions, scattered throughout the open reading frame, relative to the original sequence. A 3.7 kb GPCR/CNS mRNA species is expressed in vivo in a tissue-specific manner, with highest levels detected in brain and spinal cord, lower levels found in testis, placenta and liver, but no detectable expression observed in any other tissue. Analysis of GPCR/CNS genomic clones reveals that the human gene contains one intron that is about 21 kb in length that divides the coding region into two exons and maps to human chromosome 7q31. No specific binding is observed with either a newly identified ligand (DTyr6, beta Ala11, Phe13, Nle14]Bn-(6-14)) having high affinity for all Bn receptor subtypes or Bn after GPCR/CNS is stably expressed in fibroblasts. No elevation in inositol trisphosphate is observed after the application of micromolar levels of either DPhe6, beta Ala11, Phe13, Nle14]Bn-(6-14) or Bn, a concentration of agonist known to activate all four known Bn receptor subtypes. When GPCR/CNS is expressed in Xenopus oocytes, no activation of the calcium-dependent chloride channel is detected despite the addition of micromolar levels of Bn peptide agonists. We conclude that the natural ligand for this receptor is none of the known naturally occurring Bn-like peptides and the true agonist for GPCR/CNS remains to be elucidated.

Is a decrease in cyclic AMP a necessary and sufficient signal for maturation of amphibian oocytes

Acetylcholine rapidly lowered the intracellular levels of cyclic AMP in stage 5 and 6 Xenopus laevis oocytes. Acetylcholine alone did not induce oocyte maturation, though it did accelerate maturation induced by progesterone. The effect of acetylcholine on oocyte maturation was independent of extracellular calcium concentration. Adenosine increased cyclic AMP and abolished the progesterone-induced decrease in cyclic AMP levels in follicles and in denuded oocytes. This effect of adenosine was blocked by the Ra purinergic receptor antagonist, theophylline. Despite those effects, adenosine alone induced maturation in stage 6 oocytes and accelerated progesterone-induced maturation in both stage 5 and 6 cells. Adenosine also induced a significant increase in the rate of 45Ca efflux from oocytes in the presence and the absence of external calcium. We suggest that the activation of cell surface receptors involved in the release of calcium from cellular stores may induce or accelerate oocyte maturation independently of small changes in intracellular cyclic AMP concentration.

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.

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.

Inositol trisphosphate may access calcium from stores not coupled to muscarinic receptors in Xenopus oocytes

Oocytes of a large fraction of Xenopus females exhibit a complex response to acetylcholine (ACh) consisting of rapid, transient and prolonged, slow chloride currents. Frequent consecutive challenges or a single prolonged challenge with ACh result in a marked decrease in response amplitudes, i. e. refractoriness. In ACh-refractory oocytes, the response to injected inositol 1,4,5,-trisphosphate (InsP3,), the intracellular mediator of the ACh response, is not affected. Similarly, InsP3-evoked responses were obtained in oocytes that lacked muscarinic response or that lost their responsiveness as a result of progesterone-induced maturation. To investigate the mechanism of this phenomenon, we have depleted intracellular calcium stores by repeated challenges with ACh in calcium-free medium. Disappearance of the ACh response through depletion of the ACh-coupled calcium store did not prevent a subsequent response to InsP3. These results imply that InsP3 can mobilize calcium from other stores, not depleted by previous exposure to ACh. This finding is further reinforced by our results that demonstrate that ACh causes 45Ca efflux in responsive oocytes, while InsP3 in supramaximal concentrations does not induce 45Ca efflux. Indeed, InsP3 can induce 45Ca efflux only when more than 2 pmol/oocyte is injected. This is also the concentration of InsP3 that desensitizes the InsP3 response. These data suggest that InsP3 also releases cellular calcium from stores different from those mobilized by ACh.