A calcium influx is neither strictly associated with nor necessary for exocytotic membrane fusion in Paramecium cells.

Exocytosis of trichocysts in Paramecium cells was generally believed to depend on extracellular Ca, since it is accompanied by a Ca influx and not seen in the absence of Ca. However, by short term removal of Ca we showed recently that only extrusion of secretory contents, but not membrane fusion after stimulation with aminoethyldextran (AED), depends on extracellular Ca. We have now extended these studies to longer times and shown that membrane fusion is stimulated by AED even after 1 min at low Ca (< or = 30 nM). At prolonged times membrane fusion was induced by sole removal of Ca. In the presence of AED, trichocyst contents were slowly extruded followed by resealing of the fused membranes, indicating independency of endocytotic membrane fusion from extracellular Ca (though we observed aberrant resealing). Later on, Ca removal is followed by cell death. By using videomicroscopy, we further provide the first evidence that exocytosis is not necessarily accompanied by an influx of Ca in the presence of the usual high concentrations (1 mM), since local exocytosis at the rear end of the cells is not followed by ciliary reversal which is triggered by Ca influx. We conclude that a Ca influx is neither regularly associated with, nor necessary for, induction of exocytotic membrane fusion in Paramecium cells. As a source for a possible alternative intracellular liberation of calcium during exocytosis, we analyzed the subplasmalemmal alveolar sac system by electron spectroscopic imaging and found indications for Ca redistributions shortly after stimulation.

Functional importance of synaptobrevin and SNAP-25 during exocytosis of histamine by rat gastric enterochromaffin-like cells.

Gastric enterochromaffin-like (ECL) cells release histamine upon stimulation with gastrin in a calcium-dependent manner. The intracellular mechanisms and proteins mediating exocytosis of histamine-containing vesicles in ECL cells have not been determined yet. We used immunocytochemistry to show the localization of SNAP-25 (synaptosome-associated protein of 25 kDa) and synaptobrevin VAMP (vesicle-associated membrane protein) in ECL cells of the rat gastric mucosa and in isolated, highly enriched ECL cells, which were identified with an antibody directed against the marker enzyme histidine decarboxylase. Immunoblots of isolated ECL cells demonstrated the presence of SNAP-25, synaptobrevin, synaptophysin, synaptotagmin, and syntaxin. Histamine release from isolated ECL cells permeabilized with 8 microM digitonin (2 min) was stimulated approximately 2.5-fold upon exposure to calcium (30 microM; 10-min incubation). Preincubation with 1 microM tetanus toxin light chain for 15 min attenuated calcium-induced histamine release by 40-50% and almost completely cleaved synaptobrevin. Botulinum neurotoxin A (100 nM) totally blocked calcium-induced histamine release and cleaved SNAP-25. We conclude that synaptobrevin, synaptophysin, synaptotagmin, SNAP-25, and syntaxin are present in gastric ECL cells. Inhibition of histamine secretion by clostridial neurotoxins associated with the cleavage of synaptobrevin and SNAP-25 implicates the functional importance of these proteins in the docking and fusion of histamine vesicles.

Adrenal chromaffin cells contain functionally different SNAP-25 monomers and SNAP-25/syntaxin heterodimers.

Syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa), associated with the neuronal plasmalemma, and synaptobrevin, a membrane protein of synaptic vesicles, are essential components of the exocytotic apparatus of synaptic vesicles. All three can be proteolytically cleaved by tetanus and/or botulinum neurotoxins. As a consequence of their cleavage, exocytosis of neurotransmitters is blocked. In adrenal chromaffin cells botulinum neurotoxin A only incompletely inhibits exocytosis. This incomplete inhibition of exocytosis is associated with only partial cleavage of SNAP-25 by the toxin, indicating that distinct pools of SNAP-25 may exist in chromaffin cells which differ in their sensitivities to botulinum neurotoxin A. In line with this result we localized SNAP-25 by immunogold electron microscopy not only to the plasmalemma but also to the chromaffin vesicle membrane. Moreover, in addition to SNAP-25 monomers, stable SNAP-25/syntaxin heterodimers were found in chromaffin cells. Subfractionation studies revealed the presence of SNAP-25/syntaxin heterodimers in an enriched fraction of chromaffin vesicles. This complex proved to be stable in SDS, and SNAP-25 within heterodimers was resistant to proteolytic attack by botulinum neurotoxin A. We suggest that these preexisting heterodimers may serve as receptors of soluble NSF attachment proteins (SNAP receptors) during chromaffin vesicle exocytosis.

Functional characterization of the catalytic site of the tetanus toxin light chain using permeabilized adrenal chromaffin cells.

The molecular events underlying the inhibition of exocytosis by tetanus toxin were investigated in permeabilized adrenal chromaffin cells. We found that replacement of amino acid residues within the putative zinc binding domain of the tetanus toxin light chain such as of histidine (position 233) by cysteine or valine, or of glutamate (position 234) by glutamine completely abolished the effect of the light chains on Ca2+ induced catecholamine release. Dipicolinic acid, a strong chelating agent for zinc, also prevented the effect of the tetanus toxin light chain. Zn2+ and, less potently Cu2+ and Ni2+, but not Cd2+ and Co2+, restored the activity of the neurotoxin. These data show that zinc and the putative zinc binding domain constitute the active site of the tetanus toxin light chain. Neither captopril, an inhibitor of synaptobrevin cleavage nor peptides spanning the site of synaptobrevins cleaved by the tetanus toxin in neurons, prevented the inhibition of Ca2+ induced catecholamine release by the tetanus toxin light chain. This suggests that synaptobrevins are not a major target of tetanus toxin in adrenal chromaffin cells.

Synaptobrevin cleavage by the tetanus toxin light chain is linked to the inhibition of exocytosis in chromaffin cells.

Exocytosis of secretory granules by adrenal chromaffin cells is blocked by the tetanus toxin light chain in a zinc specific manner. Here we show that cellular synaptobrevin is almost completely degraded by the tetanus toxin light chain within 15 min. We used highly purified adrenal secretory granules to show that synaptobrevin, which can be cleaved by the tetanus toxin light chain, is localized in the vesicular membrane. Proteolysis of synaptobrevin in cells and in secretory granules is reversibly inhibited by the zinc chelating agent dipicolinic acid. Moreover, cleavage of synaptobrevin present in secretory granules by the tetanus toxin light chain is blocked by the zinc peptidase inhibitor captopril and by synaptobrevin derived peptides. Our data indicate that the tetanus toxin light chain acts as a zinc dependent protease that cleaves synaptobrevin of secretory granules, an essential component of the exocytosis machinery in adrenal chromaffin cells.

Exploring the functional domain and the target of the tetanus toxin light chain in neurohypophysial terminals.

The tetanus toxin light chain blocks calcium induced vasopressin release from neurohypophysial nerve terminals. Here we show that histidine residue 233 within the putative zinc binding motif of the tetanus toxin light chain is essential for the inhibition of exocytosis, in the rat. The zinc chelating agent dipicolinic acid as well as captopril, an inhibitor of zinc-dependent peptidases, counteract the effect of the neurotoxin. Synthetic peptides, the sequences of which correspond to motifs present in the cytoplasmic domain of the synaptic vesicle membrane protein synaptobrevin 1 and 2, prevent the effect of the tetanus toxin light chain. Our results indicate that zinc bound to the zinc binding motif constitutes the active site of the tetanus toxin light chain. Moreover they suggest that cleavage of synaptobrevin by the neurotoxin causes the inhibition of exocytotic release of vasopressin from secretory granules.

Diagnosis of symptomatic postmenopausal women by traditional Chinese medicine practitioners.

OBJECTIVE: To learn more about the way that practitioners of traditional Chinese medicine (TCM) diagnose women who have menopausal symptoms. DESIGN: We assembled a cohort of 23 postmenopausal women who had hot flushes and were otherwise healthy. Each woman was examined independently by nine practitioners of TCM on the same day. Examination consisted of medical history and physical examination. Diagnoses were recorded and counted. RESULTS: The most frequent diagnosis made by the practitioners of TCM was kidney yin deficiency, which was the diagnosis made after 168 of 207 visits (81%); 23 women seen by nine TCM practitioners. Practitioners showed good agreement regarding presence of kidney yin deficiency: in 12 women (52%), this diagnosis was made by eight of nine practitioners; in 16 women (70%), seven of nine practitioners made this diagnosis; and in all 23 women (100%), at least five of nine practitioners made this diagnosis. CONCLUSIONS: Practitioners of TCM who diagnose postmenopausal women with vasomotor symptoms are likely to make a diagnosis that includes kidney yin deficiency.

Does dong quai have estrogenic effects in postmenopausal women? A double-blind, placebo-controlled trial.

OBJECTIVE: To evaluate possible estrogenic effects of dong quai on vaginal cells and on endometrial thickness in postmenopausal women. DESIGN: Double-blind, randomized, placebo-controlled clinical trial. SETTING: Department of Obstetrics and Gynecology in a large health maintenance organization (HMO). PATIENT(S): Seventy-one postmenopausal women (mean age [+/- SD], 52.4 +/- 6 years) who had follicle-stimulating hormone levels (third-generation assay) of > 30 mIU/mL with hot flashes. INTERVENTION(S): Subjects were randomized to treatment with either dong quai or placebo for 24 weeks. MAIN OUTCOME MEASURE(S): Endometrial thickness was measured by transvaginal ultrasonography; vaginal cells were evaluated for cellular maturation; menopausal symptoms were evaluated by reviewing the Kupperman index and the diary of vasomotor flushes. RESULT(S): We observed no statistically significant differences between groups in endometrial thickness, in vaginal maturation index, in number of vasomotor flushes, or in the Kupperman index. CONCLUSION(S): Used alone, dong quai does not produce estrogen-like responses in endometrial thickness or in vaginal maturation and was no more helpful than placebo in relieving menopausal symptoms.

Diagnostic and operative arthroscopy of the knee under local anesthesia with parenteral medication.

Arthroscopy of the knee under local anesthesia was performed on 102 patients for operative and diagnostic purposes between January and December of 1985. The procedures were performed on a same day basis without a pneumatic tourniquet. The operative record of each patient was reviewed to determine postoperative diagnosis and treatment. The data was matched to a patient questionnaire that measured individual reaction to local anesthesia. The effectiveness and level of patient acceptance of this anesthetic technique for operative arthroscopy was then evaluated. Ninety-one patients responded to the questionnaire, 82 of whom had prior anesthetic experience. Ninety-five percent of the patients had minimal or no discomfort during the procedure. Eighty percent indicated a preference for local anesthesia in the event of subsequent arthroscopy. No complications relating to the anesthetic agent were noted. Arthroscopy of the knee under local anesthesia for routine operative arthroscopy was found to be safe, reliable, practical, and to have a high patient acceptance rate.

Combined ankle and subtalar instability.

Ipsilateral ankle and subtalar instability has been alluded to in the orthopaedic literature. A case demonstrating this combined instability pattern is presented and a technique for documenting this disorder is described.

Characterization of vesicular membrane-bound alpha-SNAP and NSF in adrenal chromaffin cells.

alpha-SNAP and NSF are thought to act as soluble factors, which transiently bind to a complex formed between syntaxin and SNAP-25 located at the plasma membrane and synaptobrevin at the secretory vesicle membrane, at the moment of exocytosis. Here we present data which permit the novel conclusion that alpha-SNAP and NSF are not soluble in adrenal chromaffin cells but are rather membrane-bound in particular to undocked chromaffin vesicles. Evidence for this new paradigm is derived from several experimental approaches. First, alpha-SNAP and NSF were found predominantly at cellular membranes and not in the cytosol of cracked chromaffin cells. Second, alpha-SNAP and NSF were not released from membranes by Mg2+ATP, which causes priming of vesicles. Third, immune electron microscopy and immunoblotting of chromaffin vesicles purified by immunoisolation or density gradient centrifugation revealed the presence of alpha-SNAP and NSF together with typical vesicular proteins such as synaptobrevin and synaptotagmin. In the sucrose gradient 30% alpha-SNAP and 27% NSF were recovered with chromaffin vesicles. Bound alpha-SNAP was quantified (14 molecules/vesicle), and binding was characterized with recombinant his6-tagged alpha-SNAP. Overlay blots revealed that alpha-SNAP is bound to vesicular SNAP-25 and endogenous NSF. Our data show that mature chromaffin vesicles already contain specifically bound alpha-SNAP and NSF before docking at the plasmalemma.

Compression pain in a diver with intraosseous pneumatocysts.

A 30-yr-old diver experienced pain in the area of the sacroiliac joint during the descent phase of air diving to less than 10 ATA. Computed tomography of the pelvis demonstrated two gas-filled cysts within the ilium. The mechanism by which this lesion causes pain is discussed and reports of gas within bone are reviewed.

Neuronal elements in the testis of the rhesus monkey: ontogeny, characterization and relationship to testicular cells.

Intrinsic neuron-like cells expressing the catecholamine-biosynthetic enzyme tyrosine hydroxylase (TH) were recently identified in the testis of the prepubertal rhesus monkey. In this study, we characterized the neuron-like nature of these cells and examined distribution and frequency of neuronal elements in the testes of monkeys during postnatal development, puberty and adulthood. Using immunohistochemical methods, we detected both nerve fibers and cell bodies, immunoreactive for the neuronal markers neurofilament 200 (NF-200) and synaptosomal associated protein of 25 kDa (SNAP-25), TH and neuropeptide Y (NPY) in perivascular locations, intermingled with interstitial cells and close to the wall of seminiferous tubules. Marked age-related differences in the numbers of these neuronal elements became apparent, when we quantified NF-200-immunoreactive neuronal elements. Thus, intrinsic neuron-like cell bodies were found only in the testes from immature animals (i.e. , until about 3 years of age). Conversely, nerve fibers, presumably representing mainly the extrinsic innervation, were observed at all ages although they became more prominent after the pubertal increase in LH and testosterone levels. Interestingly, another testicular cell type known to contain potent regulatory substances, mast cells, was found to be in close anatomical proximity to nerve fibers. The number of these cells, positively identified with an antibody to tryptase, increased significantly after puberty following the same pattern as nerve fibers. These results confirm that the testicular nervous system of the monkey is composed of two components, intrinsic nerve cells and extrinsic fibers, both of which are catecholaminergic and peptidergic in nature. Furthermore, both components show a marked degree of plasticity during development, especially around the time of puberty. The intratesticular locations of neuron-like cells and fibers suggest that catecholamines and neuropeptides are likely to have multiple sites of actions, and may affect Leydig cells, cells of the tubular wall and vascular cells directly and/or indirectly via intermediation of mast cells.

A cortical phosphoprotein ('PP63') sensitive to exocytosis triggering in Paramecium cells. Immunolocalization and quenched-flow correlation of time course of dephosphorylation with membrane fusion.

We had previously shown that a phosphoprotein of 63 kDa ('PP63') is rapidly and selectively dephosphorylated during synchronous (less than or equal to 1 s) trichocyst exocytosis in Paramecium cells and then rephosphorylated within less than or equal to 1 min [Zieseniss & Plattner (1985) J. Cell Biol. 101, 2028-2035]. Using a new quenched-flow device, we now find a strict correlation between PP63 dephosphorylation and the process of membrane fusion, both occurring within 80 ms. Uptake of 32P over 90 min, followed by exocytosis and rephosphorylation for 1 min, results in a rather selective phosphorylation of the dephosphorylated form, P63, to PP63. Solubilization by repeated freezing and thawing allows isolations of P63 and PP63. On isoelectric focusing autoradiograms they have pI values of 6.05, 5.95 (major spots), 5.85 and 5.75. All spots are sensitive to alkaline, but not to acidic, hydrolysis (except for the pI-6.05 spot). On two-dimensional-gel autoradiograms the most prominent spot, of pI 5.95, is most extensively de- and re-phosphorylated. This spot, from de- and re-phosphorylated samples, was used to produce monospecific antibodies. A cortical localization of PP63 was revealed by producing Western blots from isolated cell-surface fragments ('cortices') and by immunofluorescence labelling. We assume that both P63 and PP63 are attached to cortical structures, e.g. around trichocysts, though they are partly soluble. This localization and the strict correlation of PP63 dephosphorylation with exocytotic membrane fusion suggests a role in fusion regulation.

The mechanism of histamine secretion from gastric enterochromaffin-like cells.

Enterochromaffin-like (ECL) cells play a pivotal role in the peripheral regulation of gastric acid secretion as they respond to the functionally important gastrointestinal hormones gastrin and somatostatin and neural mediators such as pituitary adenylate cyclase-activating peptide and galanin. Gastrin is the key stimulus of histamine release from ECL cells in vivo and in vitro. Voltage-gated K(+) and Ca(2+) channels have been detected on isolated ECL cells. Exocytosis of histamine following gastrin stimulation and Ca(2+) entry across the plasma membrane is catalyzed by synaptobrevin and synaptosomal-associated protein of 25 kDa, both characterized as a soluble N-ethylmaleimide-sensitive factor attachment protein receptor protein. Histamine release occurs from different cellular pools: preexisting vacuolar histamine immediately released by Ca(2+) entry or newly synthesized histamine following induction of histidine decarboxylase (HDC) by gastrin stimulation. Histamine is synthesized by cytoplasmic HDC and accumulated in secretory vesicles by proton-histamine countertransport via the vesicular monoamine transporter subtype 2 (VMAT-2). The promoter region of HDC contains Ca(2+)-, cAMP-, and protein kinase C-responsive elements. The gene promoter for VMAT-2, however, lacks TATA boxes but contains regulatory elements for the hormones glucagon and somatostatin. Histamine secretion from ECL cells is thereby under a complex regulation of hormonal signals and can be targeted at several steps during the process of exocytosis.

SNAP-25 requirement for dendritic growth of hippocampal neurons.

Structure and dimension of the dendritic arbor are important determinants of information processing by the nerve cell, but mechanisms and molecules involved in dendritic growth are essentially unknown. We investigated early mechanisms of dendritic growth using mouse fetal hippocampal neurons in primary culture, which form processes during the first week in vitro. We detected a key component of regulated exocytosis, SNAP-25 (synaptosomal associated protein of 25 kDa), in axons and axonal terminals as well as in dendrites identified by the occurrence of the dendritic markers transferrin receptor and MAP2. Selective inactivation of SNAP-25 by botulinum neurotoxin A (BoNTA) resulted in inhibition of axonal growth and of vesicle recycling in axonal terminals. In addition, dendritic growth of hippocampal pyramidal and granule neurons was significantly inhibited by BoNTA. In contrast, cleavage of synaptobrevin by tetanus toxin had an effect on neither axonal nor dendritic growth. Our observations indicate that SNAP-25, but not synaptobrevin, is involved in constitutive axonal growth and dendrite formation by hippocampal neurons.