Mariann Zerbes

Histamine causes Ca2+ entry via both a storemoperated and a store-independent pathway in bovine adrenal chromaffin cells

The characteristics and properties of the increase in cytosolic [Ca2+] that occurs in bovine adrenal medullary chromaffin cells on exposure to histamine have been investigated. Specifically, these experiments were conducted to determine how much external Ca2+ enters the cell through a (capacitative) Ca2+ entry pathway activated as a consequence of intracellular Ca2+ store mobilization, relative to that which enters independently of store depletion via other channels activated by histamine. In Fura-2 loaded cells continued exposure to histamine (10 microM) caused a rapid but transient increase in cytosolic [Ca2+] followed by a lower plateau that was sustained as long as external Ca2+ was present. In the absence of external Ca2+, only the initial brief transient was observed. In cells previously treated with thapsigargin (100 nM) in Ca(2+)-free medium to deplete the internal Ca2+ stores, histamine caused no increase in cytosolic [Ca2+] when external Ca2+ was absent. Re-introduction of external Ca2+ to thapsigargin-treated store-depleted cells caused a sustained increase in cytosolic [Ca2+] that was further increased (P < 0.0002) upon exposure to histamine. The histamine-evoked increase was prevented by the H1-receptor antagonist, mepyramine (2 microM). A comparison was made between store-dependent Ca2+ entry consequent upon store mobilization with histamine in Ca(2+)-free medium and plateau phase Ca2+ entry resulting from stimulation with histamine in Ca(2+)-containing medium. The latter was found to be approximately 3 times greater in magnitude than the former (P << 0.0001) at the same concentration of histamine (10 microM). It is concluded that histamine causes Ca2+ entry not only via a capacitative entry pathway secondary to internal store mobilization, but also causes substantial Ca2+ entry through other pathways.

Isolation of serotype 1 Marek's disease viruses from vaccinated Australian flocks

Field samples were received for Mareks disease virus (MDV) isolation from clinically affected flocks from several regions of Eastern Australia. Lymphocytes were fractionated in Ficoll-Paque and passaged once or twice in chicken embryo kidney cultures. Serotype-specific virus was detected in infected cultures by indirect immunofluorescence with monoclonal antibodies. Serotype 1 MDV was isolated from 10 flocks. In samples from 5 of these flocks, serotype 2 and 3 vaccine viruses were isolated from the same specimen. In a parallel study, plasmas obtained during lymphocyte isolation were tested for antibodies to MDVs by agar gel precipitin (AGP) tests using serotype 1 and 3 antigen extracts. No correlation was observed between the rate of virus isolation and AGP positivity. The AGP test was incapable of discriminating between the different MDV serotypes.

Mobilizing Store Ca2+ in the Presence of La3+ Evokes Exocytosis in Bovine Chromaffin Cells

Abstract: The effect on exocytosis of La3+, a known inhibitor of plasma membrane Ca2+‐ATPases and Na+/Ca2+ exchangers, was studied using cultured bovine adrenal chromaffin cells. At high concentrations (0.3‐3 mM), La3+ substantially increased histamine‐induced catecholamine secretion. This action was mimicked by other lanthanide ions (Nd3+, Eu3+, Gd3+, and Tb3+), but not several divalent cations. In the presence of La3+, the secretory response to histamine became independent of extracellular Ca2+. La3+ enhanced secretion evoked by other agents that mobilize intracellular Ca2+ stores (angiotensin II, bradykinin, caffeine, and thapsigargin), but not that due to passive depolarization with 20 mM K+. La3+ still enhanced histamine‐induced secretion in the presence of the nonselective inhibitors of Ca2+‐permeant channels SKF96365 and Cd2+, but the enhancement was abolished by prior depletion of intracellular Ca2+ stores with thapsigargin. La3+ inhibited 45Ca2+ efflux from preloaded chromaffin cells in the presence or absence of Na+. It also enhanced and prolonged the rise in cytosolic [Ca2+] measured with fura‐2 during mobilization of intracellular Ca2+ stores with histamine in Ca2+‐free buffer. The results suggest that the efficacy of intracellular Ca2+ stores in evoking exocytosis is enhanced dramatically by inhibiting Ca2+ efflux from the cell.

Effect of Gd3+ on bradykinin‐induced catecholamine secretion from bovine adrenal chromaffin cells

The effects of Gd3+ on bradykinin‐ (BK‐) induced catecholamine secretion, 45Ca2+ efflux and cytosolic [Ca2+] were studied using bovine adrenal chromaffin cells. BK increased secretion in a Ca2+‐dependent manner. From 1–100 μM, Gd3+ progressively inhibited secretion induced by 30 nM BK to near‐basal levels, however from 0.3–3 mM Gd3+ dramatically enhanced BK‐induced secretion to above control levels. Gd3+ also increased basal catecholamine secretion by 2–3 fold at 1 mM. These effects were mimicked by Eu3+ and La3+. Gd3+ enhanced secretion induced by other agonists that mobilize intracellular Ca2+ stores, but simply blocked the response to K+. Gd3+ still enhanced basal and BK‐induced secretion in Ca2+‐free solution or in the presence of 30 μM SKF96365, however both effects of Gd3+ were abolished after depleting intracellular Ca2+ stores. Gd3+ (1 mM) reduced the rate of basal 45Ca2+ efflux by 57%. In Ca2+‐free buffer, BK transiently increased cytosolic [Ca2+] measured with Fura‐2. The [Ca2+] response to BK was substantially prolonged in the presence of Gd3+ (1 mM). The results suggest that Gd3+ greatly enhances the efficacy of Ca2+ released from intracellular stores in evoking catecholamine secretion, by inhibiting Ca2+ extrusion from the cytosol. This suggests that intracellular Ca2+ stores are fully competent to support secretion in chromaffin cells to levels comparable to those evoked by extracellular Ca2+ entry. Drugs that modify Ca2+ extrusion from the cell, such as lanthanide ions, will be useful in investigating the mechanisms by which intracellular Ca2+‐store mobilization couples to Ca2+‐dependent exocytosis.

A model for determining immunogenic relationships between avian infectious bronchitis viruses

SummaryA model for determining immunogenic relationships between strains of infectious bronchitis virus is described that is based upon the vaccinating dose required to induce prevention of multiplication of a standard challenge dose of an homologous strain in the lungs of specific-pathogen-free chickens. The model has been used to demonstrate that:(1)Approximately 100 times the vaccinating dose must be used to produce immunity from one compared with two doses;(2)differences of immunogenicity of >1,000-fold exist between Australian strains;(3)immunogenicity is greatest for live virus administered directly to the trachea, followed by live virus administered by the ocular route, inactivated virus administered intratracheally and inactivated virus administered intramuscularly;(4)for chickens inoculated with Australian vaccine strains, protection against challenge is unrelated to serotype.

In Situ Chelation of Ca2+ in Intracellular Stores Induces Capacitative Ca2+ Entry in Bovine Adrenal Chromaffin Cells

Abstract: Reduction of intracellular Ca2+ stores (by chelation with the EDTA structural analogue, TPEN) is itself a sufficient trigger of capacitative Ca2+ entry. TPEN may be a useful tool in studies to investigate the mechanism of capacitative Ca2+ entry, since potential confounders, such as changes in cytosolic Ca2+ and second messenger levels, are obviated and the use of pharmacological modifiers of SERCA pumps and Ca2+ release channels is avoided.

Angiotensin II Causes Calcium Entry into Bovine Adrenal Chromaffin Cells via Pathway(s) Activated by Depletion of Intracellular Calcium Stores

The characteristics and properties of the increase in cytosolic [Ca2+] that occurs in bovine adrenal medullary chromaffin cells on exposure to angiotensin II have been investigated. In fura-2 loaded cells exposure to a maximally effective concentration of angiotensin II (100 nM) caused a rapid, but transient increase in cytosolic [Ca2+] followed by a lower plateau that was sustained as long as external Ca2+ was present. In the absence of external Ca2+ only the initial brief transient was observed. In cells previously treated with thapsigargin in Ca2+-free medium to deplete the internal Ca2+ stores, angiotensin II caused no increase in cytosolic [Ca2+] when external Ca2+ was absent. Reintroduction of external Ca2+ to thapsigargin-treated, store-depleted cells caused a sustained increase in cytosolic [Ca2+] that was not further increased upon exposure to angiotensin II. Analysis of the data suggests that in bovine chromaffin cells angiotensin II causes Ca2+ entry via a pathway(s) activated as a consequence of internal store mobilization, and entry through this pathway(s) forms the majority of the sustained Ca2+ influx evoked by angiotensin II.