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PubMed1987

Human neutrophil activation with interleukin-1. A role for intracellular calcium and arachidonic acid lipoxygenation.

Human monocyte-derived interleukin-1 (IL-1) stimulated the selective extracellular release of cytoplasmic granule-associated elastase from human neutrophils. Although extracellular calcium (Ca2+) enhances but is not required for the expression of granule exocytosis, IL-1 did induce the mobilization of previously sequestered intracellular Ca2+ as measured with the highly selective fluorescent Ca2+ indicator, Quin 2. Further, IL-1 stimulated the mobilization of cell membrane-associated Ca2+ as monitored by a decrease in fluorescence of chlorotetracycline (CTC)-loaded neutrophils. W-7, a calmodulin antagonist, and TMB-8[8(N,N-diethylamino)-octyl-(3,4,5-trimethoxy)benzoate hydrochloride], an intracellular Ca2+ antagonist, inhibited the Quin 2 fluorescent response by neutrophils to IL-1. TPCK (N-alpha-p-tosyl-L-lysine chloromethylketone), a serine protease inhibitor, suppressed IL-1-induced Quin 2 and CTC fluorescence. Exposure of neutrophils to IL-1 resulted in a concentration-dependent production of the 5-lipoxygenase product, LTB4 [5(S),12(R)-dihydroxy-6,14-cis-8,10-trans-eicosatetraenoic acid] which was enhanced in the presence of arachidonic acid (AA). LTB4 production by IL-1-activated neutrophils was suppressed by the lipoxygenase inhibitors nordihydroguaiaretic acid (NDGA) and piriprost potassium [6,9,deepoxy-6,9-(phenylimino)-delta 6,8-prostaglandin l1], and a cyclooxygenase/lipoxygenase inhibitor, 5,8,11,14-eicosatetraynoic acid (ETYA), whereas a cyclooxygenase inhibitor, flurbiprofen, was inactive. These data indicate that cytosolic free Ca2+ ([Ca2+]i) and a metabolite(s) of AA lipoxygenation mediate granule exocytosis elicited with IL-1.

5,8,11,14-Eicosatetraynoic Acid↗
PubMed1987

Studies on the calcium dependence of human NK cell killing.

T lymphocytes and NK cells depend on extracellular Ca2+ to mediate cellular cytotoxicity. In the present work, we have used pharmacological tools to analyze the nature of this calcium dependence. Ca2+ channel blockers like nifedipine greater than or equal to diltiazem greater than verapamil greater than cobalt chloride inhibited NK killing but at concentrations higher than those sufficient to block voltage-operated Ca2+ channels. Quercetin and TMB-8 also suppressed killing. Depolarization or NK cells with high K+ concentration resulted in partial inhibition of lysis in contrast to hyperpolarization with K+ ionophore valinomycin which had no effect. Depolarization of hyperpolarization in the presence of a protein kinase C activator (phorbol ester, TPA) did not initiate killing of NK resistant target cells. Of the two K+ channel inhibitors tested, 4-AP and TEA, only 4-AP was inhibitory for NK killing. No release of membrane-bound Ca2+ as judged by chlorotetracycline fluorescence could be detected in the NK cell population during binding to target cells although an influx of 45Ca2+ into the NK cell population was found. Treatment of NK cells with calcium ionophore A23187 did not trigger killing, but lysis could be induced by simultaneous stimulation with A23187 and TPA. The results indicate that NK killing depends on Ca2+ channels that are different from voltage operated channels and that intracellular Ca2+ may act in concert with protein kinase C activation.

Calcimycin↗
PubMed1991

Cytosolic free Ca2+ in daunorubicin and vincristine resistant Ehrlich ascites tumor cells. Drug accumulation is independent of intracellular Ca2+ changes.

The possible role of intracellular calcium on daunorubicin (DNR) accumulation in wild-type (EHR2) and multi-drug resistant (MDR) Ehrlich ascites tumor cell subline was investigated. DNR accumulation was not enhanced either by increasing the concentration of cellular calcium with the calcium ionophore ionomycin nor by chelating the cytosolic free Ca2+ by the membrane permeable Ca2(+)-buffering agents BAPTA or MAPTAM. No effect was observed in the presence of extremely low extracellular calcium concentration that prevent transmembrane calcium influx or when the cells were calcium depleted using EGTA and ionomycin. Using the fluorescent Ca2+ indicator fura-2 it is further shown that both drug-resistant daunorubicin (EHR2/DNR+) and vincristine (EHR/VCR+) sublines had lower (50-80 nM) concentration of cytosolic free calcium ([Ca2+]i) compared to their corresponding wild-type parenteral tumors (140-180 nM). In calcium free medium, however, no significant difference was found, all cell lines having a [Ca2+]i of 60-80 nM. Furthermore, the total amount of Ca2+ released to the cytosol with 10 microM ionomycin and 5 mM EGTA was 3-4-fold higher in EHR2 than in EHR2/DNR+ or EHR2/VCR+. Mobilization of Ca2+ with 1 microM ionomycin was almost identical in the presence and absence of Ca2+ in the extracellular medium in EHR2 as well as in EHR2/DNR+ suggesting that the increase in [Ca2+]i is mainly due to discharge of Ca2+ from intracellular stores. Furthermore, the total cell calcium [Ca2+]t concentration was slightly higher in EHR2/DNR+ and EHR2/VCR+ cells compared to EHR2. Incubation of the cells with the Ca2(+)-channel blocker verapamil or the intracellular Ca2(+)-antagonist TMB-8 causes depression of the Ca2(+)-response in terms of rise in [Ca2+]i caused by ionomycin. Sorcin, a major calcium-binding protein (Mr 22 kDa), is shown to be overproduced in EHR2/DNR+ cells. The overproduction of this protein in resistant cells may be related to the difference in the intracellular calcium observed in this study. Thus, though handling of Ca2+ is different in wild-type and MDR cell lines, our data suggest that calcium is not involved directly in drug transport processes and the level of Ca2+ per se have no influence on drug accumulation.

Animals↗
PubMed1981

Cell groups in the lower brain stem of the rabbit projecting to the spinal cord, with special reference to catecholamine-containing neurons.

Two groups of experiments were carried out in rabbits. In the first groups, the distribution of cell bodies within the pons and medulla projecting ipsilaterally and contralaterally to the thoracic or lumbar spinal cord was studied using the horseradish peroxidase (HRP)/tetramethylbenzidine (TMB) procedure. In the second group, both a previously described double-labeling technique and a new modification of it were used to determine the location of catecholamine (CA)-fluorescent pontomedullary cells projecting to the spinal cord. The results demonstrate that the catecholamine (probably norepinephrine)-containing neurons which innervate the thoracic spinal cord are confirmed almost exclusively to the pons where they were found within the A5, A7 and subcoeruleus groups, as well as the ventral portion of the principal part of the locus coeruleus and the more caudal locus coeruleus, including the A4 cell group. Within the medulla oblongata no doubly labeled A2 cells were observed and the few double labeled A1 cells which were observed were confined to the rostral portion of this group. A dense group of HRP-positive but non-fluorescent cells was found rostral to the A1 area in the ventrolateral reticular formation. These cells, which correspond in position to PNMT-containing cells in the rat, appear to project to both thoracic and lumbar segments of the spinal cord. In contrast, spinally projecting neurons within the nucleus tractus solitarius originated from different subnuclei according to their segmental destination. New information about the organization of medial reticulospinal and vestibulospinal pathways was also obtained.

Animals↗
PubMed1982

Medullary proprioceptive neurons from extraocular muscles in the pigeon identified with horseradish peroxidase.

Three to five microliters of 50% HRP in saline was injected along a central axis into one of the 6 extraocular muscles in each of 18 adult pigeons. The brain was fixed and serially sectioned 16-20 h postinjection and the HRP reacted with tetramethylbenzidine (TMB). HRP-labeled proprioceptive neurons were located in the ipsilateral nucleus descendens nervi trigemini (TTD) for all muscle injections. The labeled neurons were further subdivided into two groups based on size and shape. In each experiment the number of labeled proprioceptive cells relative to the number of labeled motoneurons ranged between 4.9 and 15.5%. There were no labeled cells in the trigeminal mesencephalic nucleus or contralateral TTD. The study suggests that at least partial afferent (proprioceptive) innervation of the extraocular muscles in the pigeon derived from neurons in the ipsilateral TTD.

Animals↗
PubMed1982

Identification of multiple groups of efferent vestibular neurons in the adult pigeon using horseradish peroxidase and DAPI.

This study was designed to investigate the efferent innervation of the pigeon labyrinth. Horseradish peroxidase (HRP) was injected and confined within the endolymphatic space of one labyrinth in 9 adult pigeons. The brain was perfused by transcardiac carotid catheterization and the HRP reacted by the tetramethylbenzidine (TMB) blue reaction process. Five different groups of HRP-labeled vestibular efferent neurons were identified. Three groups were located within the confines of the ipsilateral vestibular nuclear complex (in the lateral, tangential and descending nucleus) and two additional groups, each bilateral, were located in the reticular formation. In 9 additional pigeons, 4,6-diamidino-2-phenylindole (DAPI) was also injected and confined within the endolymphatic space of one labyrinth. DAPI-labeled cells were noted in 3 of the 5 locations (tangential nucleus, and both reticular groups) which in the other animals contained HRP-labeled cells. These findings raise the possibility of different physiological roles for the efferent vestibular groups in the ipsilateral vestibular nuclear complex and bilateral reticular formation.

Animals↗
PubMed1982

Afferent connections of the ectostriatum and visual wulst in the zebra finch (Taeniopygia guttata castanotis Gould)--an HRP study.

Afferent connections of the two main areas in the telencephalon, the visual wulst and the ectostriatum, were traced in the zebra finch by injection of horseradish peroxidase and staining with tetramethylbenzidine (TMB). Nuclei projecting to the hyperstriatum accessorium (HA) or the HIS region (lamina hyperstriatica intercalatus superior) were: (1) ipsilaterally the n. dorsalis anterior pars lateralis (DLL) with its two subdivisions DLLd and DLLv, the n. dorsolateralis anterior pars magnocellularis (DLAmc), and the area pretectalis (AP); (2) bilaterally the nucleus of the septomesencephalic tract (SPC) with the ipsilateral component coming from the medial, the contralateral component from the lateral part of the nucleus. As in the pigeon or the owl the ectostriatum of the zebra finch receives massive input, which is topographically ordered, from the n. rotundus. In addition to this pathway the ectostriatum receives additional visual input from the ipsilateral area pretectalis, the n. subrotundus and eventually a bilateral projection from the n. tegmenti pedunculopontinus pars compacta (TPC).

Animals↗
PubMed1984

Retrograde double labeling of neurons: the combined use of horseradish peroxidase and diamidino yellow dihydrochloride (DY X 2HCl) compared with true blue and DY X 2HCl in rat descending brainstem pathways.

Three series of double-labeling experiments were carried out in a study of the collateralization of brainstem nuclei which project to the spinal cord in the rat. The fluorescent tracer Diamidino Yellow Dihydrochloride (DY X 2HCl) was injected in one half of the spinal gray and white matter at T7-T8 or T13-L1. Subsequently, either True Blue (TB), wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) or free HRP were injected ipsilaterally in the gray matter at C5-C8. The distributions of single and double retrogradely labeled neurons were studied in the following cell groups: red nucleus, interstitial nucleus of Cajal, ventrolateral pontine tegmentum, nuclei coeruleus and subcoeruleus and nuclei raphe magnus and raphe pallidus including the adjoining ventral reticular formation. The numbers of TB- or HRP-labeled neurons present in those nuclei were counted and the percentages of double-labeled neurons were calculated when TB or HRP had been used in combination with DY X 2HCl. The results indicate: The HRP-TMB reaction product and the DY X 2HCl fluorescence can be visualized simultaneously in retrogradely single- or double-labeled neurons. The distributions of single- and double-labeled neurons in the various brainstem nuclei were entirely comparable when using TB with DY X 2HCl or HRP with DY X 2HCl. The percentages of double-labeled neurons obtained with HRP and DY X 2HCl were consistent over a series of cases, and were comparable to those obtained with TB and DY X 2HCl in several structures. However, in the red nucleus slightly lower percentages of double-labeled neurons were obtained using HRP and DY X 2HCl as compared with the percentages obtained using TB and DY X 2HCl.

Amidines↗
PubMed1985

ATP-dependent [3H]Met-enkephalin uptake by bovine adrenal chromaffin granule membrane.

Uptake of [3H]Met-enkephalin by purified chromaffin granule membrane isolated from bovine adrenal medulla was investigated. Addition of a single divalent cation, such as Mg2+ or Ca2+, did not affect the uptake. The presence of 3 mM ATP in the incubation medium stimulated the uptake to two times that of the control. The same stimulation effect was also found in the presence of ATP plus a single divalent cation. The ATP-dependent uptake reached its half-maximal level within 5 min after initiation of the reaction at 25 degrees C, and reached a plateau within 10 min. The apparent Km for [3H]Met-enkephalin uptake by the chromaffin granule membrane was about 2.3 X 10(-6) M. GTP, CTP, UTP and ADP did not stimulate enkephalin uptake. Several calcium inhibitors such as trifluoperazine, verapamil and 3,4,5-trimethoxybenzoic acid 8-(N,N-diethylamino)octylester (TMB-8) markedly inhibited enkephalin uptake. The inhibition exceeded 80%, although these chemicals inhibited Ca2+-ATPase activity in chromaffin granule membrane only 30% under the same conditions. Ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid (EGTA), at 3 mM also inhibited uptake about 30%. The results indicate that uptake of Met-enkephalin by chromaffin granule membrane ghosts was driven not only by ATPase activity, but also by some other Ca2+-ATP-mediated mechanism(s).

Adenosine Triphosphate↗
PubMed1988

Prostaglandin E2 releases luteinizing hormone-releasing hormone from the female juvenile hypothalamus through a Ca2+-dependent, calmodulin-independent mechanism.

Prostaglandin E2 (PGE2) has been implicated as a mediator of norepinephrine-induced luteinizing hormone-releasing hormone (LHRH) release from the hypothalamus. The present experiments were undertaken to examine the hypothesis that mobilization of Ca2+ from intracellular stores and cyclic AMP (cAMP) formation are involved in this effect. Incubation of median eminence (ME) nerve terminals from juvenile rats in Ca2+-free Krebs-Ringer bicarbonate buffer reduced, but failed to prevent the stimulatory effect of PGE2 on LHRH release. None of 5 calmodulin antagonists or a blocker of calmodulin-dependent kinase affected the LHRH response to PGE2. In contrast, inhibition of intracellular Ca2+ mobilization with TMB-8 or dantrolene in Ca2+-free medium prevented the LHRH releasing effect of PGE2. Similarly to PGE2, the stimulatory effect of the Ca2+ ionophore A 23187 on LHRH release was not affected by inhibition of calmodulin activity. This, however, blocked the increase in PGE2 formation induced by the ionophore. PGE2 evoked a dose-related increase in cAMP accumulation in Ca2+-containing medium and this effect was inhibited both by blockers of intracellular Ca2+ mobilization and by calmodulin antagonists. Surprisingly, removal of extracellular Ca2+ increased basal cAMP levels in the incubation medium without affecting LHRH release; PGE2 induced a further increase in cAMP which was prevented by inhibition of intracellular Ca2+ translocation. Stimulation of adenylate cyclase activity with forskolin (F) resulted in similar increases in cAMP levels both in the presence and absence of extracellular Ca2+. However, F failed to evoke LHRH release in Ca2+-free medium. The results indicate that: (a) the stimulatory effect of PGE2 on LHRH release involves mobilization of intracellular Ca2+ but not the participation of calmodulin; (b) the formation of PGE2 itself is calmodulin-dependent; (c) PGE2 stimulates cAMP formation through a calmodulin-dependent mechanism that requires translocation of intracellular (membrane?) Ca2+; (d) the cAMP system of a population of nerve terminals different from that responsive to PGE2 is normally subjected to a Ca2+-dependent inhibitory control; and (e) although PGE2 is a potent stimulator of cAMP formation in the ME and endogenously produced cAMP can induce LHRH release, in the absence of extracellular Ca2+ PGE2 stimulates LHRH release in a cAMP-independent manner.

Animals↗
PubMed1992

Organization of cutaneous primary afferent fibers projecting to the dorsal horn in the rat: WGA-HRP versus B-HRP.

Primary afferent projections from cutaneous afferents in the forelimb and hindlimb digits to the dorsal horn (DH) were examined using 4 tracers: (1) 25% free horseradish peroxidase (HRP), (2) 2.5% wheat-germ agglutinin conjugated to horseradish peroxidase (WGA-HRP), (3) a mixture of 25% free HRP and 2.5% WGA-HRP (WGA-HRP/HRP) or (4) 0.1% HRP conjugated to cholera toxin (B-HRP). The tracer was injected intracutaneously into the digits. Three to 4 days later, the rats were perfused transcardially, transverse sections (60-microns thick) were cut and the HRP was reacted using the tetramethyl benzidine (TMB) method. The location of the label was reconstructed by camera lucida drawings. In rats which received an injection of HRP alone, no label was detected in the DH. Rats injected with WGA-HRP had projection patterns similar to those injected with WGA-HRP/HRP. Patterns of labelling with WGA-HRP differed markedly from those with B-HRP. WGA-HRP labelled cutaneous afferents projecting to Rexed's laminae I-III, with the densest label in lamina II; in contrast, B-HRP labelled cutaneous afferents projecting to laminae II-V, with the densest label in laminae III-IV. These results indicate that, for cutaneous primary afferents projecting to the DH, WGA-HRP and B-HRP labelled different subpopulations of fibers, with the B-HRP-labelled subpopulation biased toward afferents of larger diameter. Rostrocaudally, the extent of the densest fiber projections, whether labelled by WGA-HRP or by B-HRP, was essentially the same, but the extent of the less densely labelled projections was much greater with B-HRP than with WGA-HRP. Comparisons of the projection maps from each of the five digits, using either WGA-HRP or B-HRP, indicated that, as seen in transverse sections through the DH, there was extensive overlapping among the labelled cutaneous afferent fibers from adjacent, or even non-adjacent digits.

Afferent Pathways↗
PubMed1992

Dopamine receptor antagonists increase markedly the quantity of retrograde transport of HRP in the rat masseteric motoneuron.

Horseradish peroxidase (HRP) was injected, bilaterally, into the rat masseter muscle, subsequent to an intramuscular or intraperitoneal injection of one of five dopamine antagonists (chlorpromazine and haloperidol as the D1 and D2 receptor antagonist, SCH 23390 as the specific D1 receptor antagonist, sulpiride and domperidone as the specific D2 receptor antagonist). Control rats received an injection of a corresponding vehicle solution. After a survival period of 16 h, the brainstem was cut into 60 microns cryosections and processed with the TMB technique. The amount of retrogradely transported HRP was quantitatively measured in terms of the amount of HRP reaction product present in the motoneuron by the method which we have developed using an image processing system combined with a light microscope and a TV camera. Chlorpromazine, haloperidol, SCH 23390 and sulpiride significantly raised the quantity of retrograde transport of HRP. On the contrary, domperidone which can not penetrate the blood-brain barrier showed no significant change in the amount of the retrograde transport. In addition, an intravenous injection of chlorpromazine (8 mg/kg) was found to increase the amplitude of monosynaptic masseteric reflex EMG activity evoked by stimulations of the mesencephalic trigeminal nucleus. These results suggest that a possible regulatory system involving the dopamine receptor in the uptake and retrograde transport of HRP from axon terminals to cell bodies of the masseteric motoneuron exists in higher order neurons which make synaptic contact with the motoneuron.

Animals↗
PubMed1995

Time course of reinnervation of the olfactory bulb after transection of the primary olfactory nerve in the pigeon.

Horseradish peroxidase (HRP) histochemistry was used to study the time course of reinnervation of the pigeon olfactory bulb after simple transection of the primary olfactory nerve. At selected intervals (9, 13, 29, 61 and 93 days) after transection of the right olfactory nerve, a concentrated solution of HRP was instilled in both nasal cavities. Intracarotid perfusion was performed 3 days after nasal instillation of HRP and 40-microns sections of olfactory bulb processed with the tetramethylbenzidine (TMB)-HRP histochemical protocol to visualize olfactory receptor axon terminals reinnervating the glomerular layer of the bulb. The total area of reinnervation of four representative regions in the bulb of the transected side were compared with that on the control bulb. The area of innervation by newly reconstituted olfactory axons was approximately 17% of control values at the 12-day posttransection time interval. A progressive increase in the area of reinnervation was observed over time. Reinnervation of the right bulb was approximately 70% complete at the 32-day posttransection time interval and indistinguishable from the left control bulb at the 64- and 96-day posttransection time intervals. A uniform pattern of reinnervation of different bulb regions was observed at all time intervals. These results indicate that the peripheral olfactory system of the pigeon is capable of complete reconstitution after nerve transection. Our findings should be useful in guiding functional comparisons of normal and newly reconstituted peripheral olfactory systems in the pigeon.

Animals↗
PubMed1990

Signal transduction mechanisms of Ia induction in B cells by interleukin 4 and immunoglobulin receptors.

Molecular mechanisms of signal transduction through receptors for interleukin 4 (IL-4) are still largely unknown. To elucidate the second messenger(s) of IL-4 action in mature B cells, we performed blocking experiments with inhibitors of various aspects of cellular responses, using Ia-inducing activity of IL-4 as a readout system. In the event, only agents that are shown to inhibit calcium ion (Ca2+) release from intracellular stores, such as 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8) and ryanodine, could block the IL-4 action in a dose-dependent fashion. These results suggest that the process leading to the final expression of IL-4 action may be mediated, at some point, by the release of Ca2+ from intracellular stores. In the parallel experiments with antiimmunoglobulin (Ig) antibody, we found that amiloride, an inhibitor of Na+/H+ pump, blocks the Ia induction by anti-IgM antibody. Thus the Na+/H+ exchange system activated by anti-Ig antibody may be present in mature B cells.

Animals↗
PubMedDate not supplied

Neuropsychological screening in the psychiatric emergency room.

In a pilot study, a neuropsychological minibattery of tests consisting of Trail-Making Test A (TMA), Trail-Making Test B (TMB), and the Visual Reproduction subtest (VR) of the Wechsler Memory Scale was administered to patients with common psychiatric diagnoses in a psychiatric emergency room (ER). Patients with adjustment disorders were not distinguishable from normal controls, while patients with affective disorders and schizophrenia were more impaired than both of these samples. It is suggested that this or a similar minibattery of tests can be of use as an adjunct screening device for differential diagnosis of adjustment disorder versus more serious psychopathology in psychiatric ERs.

Adjustment Disorders↗
PubMed1985

The wave of activation current in the Xenopus egg.

A ring-shaped wave of inward current, the activation current, propagates across the Xenopus egg from the site of activation during the positive phase of the activation or fertilization potential. This activation current wave is due to an increased chloride conductance and reflects the propagated of the ionic channels responsible for the fertilization potential. These channels are present in the animal and vegetal hemispheres; however, the magnitude of the activation current is 6-7 times greater in the animal hemisphere. Outward current of a smaller magnitude and spread out over a larger area precedes and follows the inward current except at the point of activation where the current is first inward. The inward current wave is detected in all eggs activated by sperm and in eggs activated by pricking with a sharp needle, by application of the Ca2+ ionophore, A23187, and by intracellular iontophoresis of Ca2+ or inositol 1,4,5-trisphosphate. Reduction of the inward current by TMB-8, which blocks intracellular calcium release in some cells, suggests that the activation current channels are calcium sensitive and that the current wave is concomitant with a wave of increased intracellular calcium initiated by sperm-egg interaction. The wave of cortical granule exocytosis and two or more contraction waves follow the current wave.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1987

A rise in cytosolic calcium is not necessary for maturation of Xenopus laevis oocytes.

Cytoplasmic free calcium levels during progesterone-induced meiotic maturation in Xenopus laevis oocytes were measured using the photoprotein aequorin. The resting level of [Ca2+]i was 92.6 +/- 30 nM. No significant changes were observed after progesterone addition, although a large pulse of [Ca2+]i was observed upon activation of matured oocytes. These findings are discussed in terms of the role of calcium in maturation and it is concluded that calcium is not the second messenger for progesterone. This conclusion is further supported by the finding that 100 microM TMB-8, a blocker of intracellular calcium release, had no effect on progesterone-induced maturation.

Aequorin↗
PubMed1988

The cortical reaction in the egg of Discoglossus pictus: a study of the changes in the endoplasmic reticulum at activation.

In Discoglossus pictus previous ultrastructural observations have shown that at the animal dimple, where sperm fuse with the egg, cortical granules (CG), vacuoles, and tightly packed clusters of small cisternae are present. At fertilization the clusters open (i.e., become loose) and give rise to longer cisternae arranged in whorls and chains which migrate toward the plasma membrane. The vacuoles fuse to form cisternae and exocytose along with the CG. In the rest of the egg periphery, while exocytosis occurs, the clusters do not open as a result of activation (C. Campanella, R. Talevi, U. Atripaldi, and L. Quaglia (1986). In "Molecular and Cellular Biology of Fertilization" (J.L. Hedrick, Ed.). Plenum, New York). We have recently conducted electrophysiological studies which have detected inward currents at the dimple center, outward current at the rest of the egg surface, and an eightfold increase in [Ca2+]i which propagates from the site of activation throughout the egg (R. Nuccitelli, D. Kline, W. Busa, R. Talevi, and C. Campanella (1988). Dev. Biol. 130, 120-132). In this paper we have asked whether the anionic current and the Ca2+ increase could be causally related to the changes of the smooth endoplasmic reticulum (SER) at activation. The results obtained by activating the eggs in ion-substituted Ringers indicate that (1) the migration of cisternae is not dependent on the polarity of the activation current crossing the dimple, but is strongly impaired, together with CG exocytosis, by 5 x Cl- Ringer; (2) TMB-8, a drug which partially blocks calcium release (C. Y. Choiu and M. J. Malagodi (1975). Brit. J. Pharmacol. 53, 279-288), partially inhibits opening of cisternae clusters and the formation of an SER network in the dimple. This suggests a causal relationship between the Ca2+ rise and the cluster transformation at activation.

Animals↗