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B Hille

Publications and source records attributed to B Hille.

At least 19 recordsLinked to original sources
PubMed2004

Simplification of rat intubation on inclined metal plate.

Small-animal intubation is often necessary during inhalation anesthesia to allow steady-state conditions for large operations and in vivo experiments in all fields of experimental surgery. In rats, placing an orotracheal tube is technically difficult primarily because of the small size of the subject and the lack of equipment specifically designed for this task. We describe a simple rat intubation technique in which the animal is suspended in dorsal recumbency on an inclined metal plate. The animal, anesthetized with ether, is fixed to a 70 degrees-inclined metal plate in a dorsal position by means of a Mersilene ribbon hooked around the upper incisors. This method of positioning the animal is the most important step in the intubation process and further facilitates the technique already described by other authors. A human otoscope was used as a laryngoscope, intubation was performed using the Seldinger technique, and a 14-gauge intravenous catheter served as an endotracheal tube. This inexpensive technique is quickly learned and can be used in any laboratory. Safe and reliable airway management can thus be achieved, permitting in vivo examinations and operations.

Anesthetics, Inhalation↗
PubMed2001

Identification of subtypes of muscarinic receptors that regulate Ca2+ and K+ channel activity in sympathetic neurons.

Many different G protein-coupled receptors modulate the activity of Ca2+ and K+ channels in a variety of neuronal types. There are five known subtypes (M1-M5) of muscarinic acetylcholine receptors. Knockout mice lacking the M1, M2, or M4 subtypes are studied to determine which receptors mediate modulation of voltage-gated Ca2+ channels in mouse sympathetic neurons. In these cells, muscarinic agonists modulate N- and L-type Ca2+ channels and the M-type K+ channel through two distinct, G-protein mediated pathways. The fast and voltage-dependent pathway is lacking in the M2 receptor knockout mice. The slow and voltage-independent pathway is absent in the M1 receptor knockout mice. Neither pathway is affected in the M4 receptor knockout mice. Muscarinic modulation of the M current is absent in the M1 receptor knockout mice, and can be reconstituted in a heterologous expression system using cloned channels and M1 receptors. Our results using knockout mice are compared with pharmacological data in the rat.

Animals↗
PubMed2000

Loading of oxidizable transmitters into secretory vesicles permits carbon-fiber amperometry.

Carbon-fiber amperometry detects oxidizable molecules released by exocytosis. We extended this electrochemical technique to cells that do not normally secrete oxidizable transmitters. We incubated AtT-20 cells, pituitary gonadotropes, cultured cerebellar granule cells, and yeast with high concentrations of dopamine (DA) and observed spontaneous and evoked quantal release of DA by amperometry. The rate of detectable spontaneous amperometric events was used as a measure of loading in AtT-20 cells. With 70 mm DA in the bath, loading was complete within 40 min. Cytoplasmic accumulation preceded vesicular loading. Loading decreased proportionally as the bath DA concentration was lowered. Loading rates were similar at 37 and 25 degrees C and much slower at 15 degrees C. Loading was blocked by bafilomycin A(1), a proton pump inhibitor, but not by bupropion, an inhibitor of the plasma membrane DA transporter. Other cells were tested. Spontaneous quantal events became more frequent and evoked events became larger and more frequent when PC12 cells were loaded with DA. Fluid-phase loading of neurons by short stimulation in DA solutions seemed selective for the synaptic vesicles. Thus, many cell types can be loaded with DA to study spontaneous and evoked exocytosis. The amine molecules enter these cells passively and may become concentrated in acidic vesicles by protonation.

Action Potentials↗
PubMed2000

Mitochondria shape hormonally induced cytoplasmic calcium oscillations and modulate exocytosis.

Pituitary gonadotropes transduce hormonal input into cytoplasmic calcium ([Ca(2+)](cyt)) oscillations that drive rhythmic exocytosis of gonadotropins. Using Calcium Green-1 and rhod-2 as optical measures of cytoplasmic and mitochondrial free Ca(2+), we show that mitochondria sequester Ca(2+) and tune the frequency of [Ca(2+)](cyt) oscillations in rat gonadotropes. Mitochondria accumulated Ca(2+) rapidly and in phase with elevations of [Ca(2+)](cyt) after GnRH stimulation or membrane depolarization. Inhibiting mitochondrial Ca(2+) uptake by the protonophore CCCP reduced the frequency of GnRH-induced [Ca(2+)](cyt) oscillations or, occasionally, stopped them. Much of the Ca(2+) that entered mitochondria is bound by intramitochondrial Ca(2+) buffering systems. The mitochondrial Ca(2+) binding ratio may be dynamic because [Ca(2+)](mit) appeared to reach a plateau as mitochondrial Ca(2+) accumulation continued. Entry of Ca(2+) into mitochondria was associated with a small drop in the mitochondrial membrane potential. Ca(2+) was extruded from mitochondria more slowly than it entered, and much of this efflux could be blocked by CGP-37157, a selective inhibitor of mitochondrial Na(+)-Ca(2+) exchange. Plasma membrane capacitance changes in response to depolarizing voltage trains were increased when CCCP was added, showing that mitochondria lower the local [Ca(2+)](cyt) near sites that trigger exocytosis. Thus, we demonstrate a central role for mitochondria in a significant physiological response.

Animals↗
PubMed2000

CaV2.2 and CaV2.3 (N- and R-type) Ca2+ channels in depolarization-evoked entry of Ca2+ into mouse sperm.

As sperm prepare for fertilization, surface Ca(2+) channels must open to initiate required, Ca(2+)-mediated events. However, the molecular identity and functional properties of sperm Ca(2+) channels remain uncertain. Here, we use rapid local perfusion and single-cell photometry to examine the kinetics of calcium responses of mouse sperm to depolarizing stimuli. The linear rise of intracellular [Ca(2+)] evoked by approximately 10-s applications of an alkaline high [K(+)] medium directly reports activity of voltage-gated Ca(2+) channels. Little response occurs if external Ca(2+) is removed or if external or internal pH is elevated without depolarization. Responses are inhibited 30-40% by 30-100 micrometer Ni(2+) and more completely by 100-300 micrometer Cd(2+). They resist the dihydropyridines nitrendipine and PN200-110, but 1-10 micrometer mibefradil inhibits reversibly. They also resist the venom toxins calciseptine, omega-conotoxin MVIIC, and kurtoxin, but omega-conotoxin GVIA (5 micrometer) inhibits approximately 50%. GVIA also partially blocks transient, low voltage activated Ca(2+) currents of patch-clamped spermatids. Differential sensitivity of sperm responses to Ni(2+) and Cd(2+) and partial blockade by GVIA indicate that depolarization opens at least two types of voltage-gated Ca(2+) channels in epididymal sperm examined prior to capacitation. Involvement of a previously undetected Ca(V)2.2 (N-type) channel, suggested by the action of GVIA, is substantiated by immunodetection of Ca(2+) channel alpha(1B) subunits in sperm and sperm extracts. Resistance to dihydropyridines, calciseptine, MVIIC, and kurtoxin indicates that Ca(V)1, Ca(V)2.1, and Ca(V)3 (L-, P/Q-, and T-type) channels contribute little to this evoked response. Partial sensitivity to 1 micrometer mibefradil and an enhanced sensitivity of the GVIA-resistant component of response to Ni(2+) suggest participation of a Ca(V)2.3 (R-type) channel specified by previously found alpha(1E) subunits. Our examination of depolarization-evoked Ca(2+) entry indicates that mature sperm possess a larger palette of voltage-gated Ca(2+) channels than previously thought. Such diversity may permit specific responses to multiple cues encountered on the path to fertilization.

Animals↗
PubMed2000

Reconstitution of muscarinic modulation of the KCNQ2/KCNQ3 K(+) channels that underlie the neuronal M current.

Channels from KCNQ2 and KCNQ3 genes have been suggested to underlie the neuronal M-type K(+) current. The M current is modulated by muscarinic agonists via G-proteins and an unidentified diffusible cytoplasmic messenger. Using whole-cell clamp, we studied tsA-201 cells in which cloned KCNQ2/KCNQ3 channels were coexpressed with M(1) muscarinic receptors. Heteromeric KCNQ2/KCNQ3 currents were modulated by the muscarinic agonist oxotremorine-M (oxo-M) in a manner having all of the characteristics of modulation of native M current in sympathetic neurons. Oxo-M also produced obvious intracellular Ca(2+) transients, observed by using indo-1 fluorescence. However, modulation of the current remained strong even when Ca(2+) signals were abolished by the combined use of strong intracellular Ca(2+) buffers, an inhibitor of IP(3) receptors, and thapsigargin to deplete Ca(2+) stores. Muscarinic modulation was not blocked by staurosporine, a broad-spectrum protein kinase inhibitor, arguing against involvement of protein kinases. The modulation was not associated with a shift in the voltage dependence of channel activation. Homomeric KCNQ2 and KCNQ3 channels also expressed well and were modulated individually by oxo-M, suggesting that the motifs for modulation are present on both channel subtypes. Homomeric KCNQ2 and KCNQ3 currents were blocked, respectively, at very low and at high concentrations of tetraethylammonium ion. Finally, when KCNQ2 subunits were overexpressed by intranuclear DNA injection in sympathetic neurons, total M current was fully modulated by the endogenous neuronal muscarinic signaling mechanism. Our data further rule out Ca(2+) as the diffusible messenger. The reconstitution of muscarinic modulation of the M current that uses cloned components should facilitate the elucidation of the muscarinic signaling mechanism.

Animals↗
PubMed2000

Regulation of exocytosis by protein kinases and Ca(2+) in pancreatic duct epithelial cells.

We asked if the mechanisms of exocytosis and its regulation in epithelial cells share features with those in excitable cells. Cultured dog pancreatic duct epithelial cells were loaded with an oxidizable neurotransmitter, dopamine or serotonin, and the subsequent release of these exogenous molecules during exocytosis was detected by carbon-fiber amperometry. Loaded cells displayed spontaneous exocytosis that may represent constitutive membrane transport. The quantal amperometric events induced by fusion of single vesicles had a rapid onset and decay, resembling those in adrenal chromaffin cells and serotonin-secreting leech neurons. Quantal events were frequently preceded by a "foot," assumed to be leak of transmitters through a transient fusion pore, suggesting that those cell types share a common fusion mechanism. As in neurons and endocrine cells, exocytosis in the epithelial cells could be evoked by elevating cytoplasmic Ca(2+) using ionomycin. Unlike in neurons, hyperosmotic solutions decreased exocytosis in the epithelial cells, and giant amperometric events composed of many concurrent quantal events were observed occasionally. Agents known to increase intracellular cAMP in the cells, such as forskolin, epinephrine, vasoactive intestinal peptide, or 8-Br-cAMP, increased the rate of exocytosis. The forskolin effect was inhibited by the Rp-isomer of cAMPS, a specific antagonist of protein kinase A, whereas the Sp-isomer, a specific agonist of PKA, evoked exocytosis. Thus, PKA is a downstream effector of cAMP. Finally, activation of protein kinase C by phorbol-12-myristate-13-acetate also increased exocytosis. The PMA effect was not mimicked by the inactive analogue, 4alpha-phorbol-12,13-didecanoate, and it was blocked by the PKC antagonist, bisindolylmaleimide I. Elevation of intracellular Ca(2+) was not needed for the actions of forskolin or PMA. In summary, exocytosis in epithelial cells can be stimulated directly by Ca(2+), PKA, or PKC, and is mediated by physical mechanisms similar to those in neurons and endocrine cells.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1999

Stimulation of exocytosis without a calcium signal.

More than 30 years ago, Douglas (Douglas & Rubin, 1961; Douglas, 1968) proposed that intracellular Ca2+ controls stimulus-secretion coupling in endocrine cells, and Katz & Miledi (1967; Katz, 1969) proposed that intracellular Ca2+ ions control the rapid release of neurotransmitters from synapses. These related hypotheses have been amply confirmed in subsequent years and for students of excitable cells, they dominate our teaching and research. Calcium controls regulated exocytosis. On the other hand, many studies of epithelial and blood cell biology emphasize Ca2+-independent regulation of secretion of mucin, exocytotic delivery of transporters and degranulation. The evidence seems good. Are these contrasting conclusions somehow mistaken, or are the dominant factors controlling exocytosis actually different in different cell types? In this essay, we try to reconcile these ideas and consider classes of questions to ask and hypotheses to test in seeking a more integrated understanding of excitation-secretion coupling. Our review is conceptual and narrowly selective of a few examples rather than referring to a broader range of useful studies in the extensive literature. The examples are taken from mammals and are documented principally by citing other reviews and two of our own studies. The evidence shows that protein phosphorylation by kinases potentiates Ca2+-dependent exocytosis and often suffices to induce exocytosis by itself. Apparently, protein phosphorylation is the physiological trigger in a significant number of examples of regulated exocytosis. We conclude that although sharing many common properties, secretory processes in different cells are specialized and distinct from each other.

Animals↗
PubMed1999

Assignment of muscarinic receptor subtypes mediating G-protein modulation of Ca(2+) channels by using knockout mice.

There are five known subtypes of muscarinic receptors (M(1)-M(5)). We have used knockout mice lacking the M(1), M(2), or M(4) receptors to determine which subtypes mediate modulation of voltage-gated Ca(2+) channels in mouse sympathetic neurons. Muscarinic agonists modulate N- and L-type Ca(2+) channels in these neurons through two distinct G-protein-mediated mechanisms. One pathway is fast and membrane-delimited and inhibits N- and P/Q-type channels by shifting their activation to more depolarized potentials. The other is slow and voltage-independent and uses a diffusible cytoplasmic messenger to inhibit both Ca(2+) channel types. Using patch-clamp methods on acutely dissociated sympathetic neurons, we isolated each pathway by pharmacological and kinetic means and found that each one is nearly absent in a particular knockout mouse. The fast and voltage-dependent pathway is lacking in the M(2) receptor knockout mice; the slow and voltage-independent pathway is absent from the M(1) receptor knockout mice; and neither pathway is affected in the M(4) receptor knockout mice. The knockout effects are clean and are apparently not accompanied by compensatory changes in other muscarinic receptors.

Animals↗
PubMed1999

Rapid fabrication of plastic-insulated carbon-fiber electrodes for micro-amperometry.

Carbon-fiber amperometry and voltammetry are useful techniques to measure secretion of oxidizable neurotransmitters from neurosecretory cells. Recent applications with probes of small geometry permit detection of the exocytosis of single secretory vesicles in individual cells. We have developed a semi-automatic puller and cutter to prepare such plastic-insulated electrodes efficiently with various sizes of carbon fibers. The electrodes are smooth, reproducible, and easy to make.

Amines↗
PubMed1999

Distribution and function of angiotensin II receptors in mouse spermatozoa.

Previous work indicates that angiotensin II (AngII) stimulates sperm motility and acrosomal exocytosis. Here we determined the distribution of AngII receptors on mouse sperm by immunocytochemistry and used Ca2+ probe photometry to examine their coupling to sperm regulatory pathways. We found both AT1 and AT2 receptors localized on the acrosomal region of the sperm head. The AT1 receptor, but not the AT2 receptor, is found also on the principal piece of the sperm tail. Local perfusion of motile but nonprogressive sperm with 0.1-1 microM of AngII evokes a rapid, substantial rise in intracellular [Ca2+]. This response is blocked by losartan, a specific antagonist of the AT1 receptor. These results indicate that sperm possess functional AT1 receptors that are distributed to sites that may allow selective control of motility and exocytosis. They also suggest that the AT2 receptors detected by immunoreactivity are either nonfunctional or are not coupled to Ca(2+)-mediated signalling mechanisms.

Angiotensin II↗
PubMed1998

G-protein beta-subunit specificity in the fast membrane-delimited inhibition of Ca2+ channels.

We investigated which subtypes of G-protein beta subunits participate in voltage-dependent modulation of N-type calcium channels. Calcium currents were recorded from cultured rat superior cervical ganglion neurons injected intranuclearly with DNA encoding five different G-protein beta subunits. Gbeta1 and Gbeta2 strongly mimicked the fast voltage-dependent inhibition of calcium channels produced by many G-protein-coupled receptors. The Gbeta5 subunit produced much weaker effects than Gbeta1 and Gbeta2, whereas Gbeta3 and Gbeta4 were nearly inactive in these electrophysiological studies. The specificity implied by these results was confirmed and extended using the yeast two-hybrid system to test for protein-protein interactions. Here, Gbeta1 or Gbeta2 coupled to the GAL4-activation domain interacted strongly with a channel sequence corresponding to the intracellular loop connecting domains I and II of a alpha1 subunit of the class B calcium channel fused to the GAL4 DNA-binding domain. In this assay, the Gbeta5 subunit interacted weakly, and Gbeta3 and Gbeta4 failed to interact. Together, these results suggest that Gbeta1 and/or Gbeta2 subunits account for most of the voltage-dependent inhibition of N-type calcium channels and that the linker between domains I and II of the calcium channel alpha1 subunit is a principal receptor for this inhibition.

Adrenergic Fibers↗
PubMed1998

Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons.

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B2 bradykinin and M1 muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP3 receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP3 to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca2+]i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca2+]i were blocked by depletion of Ca2+ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca2+ from IP3-sensitive Ca2+ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.

Animals↗
PubMed1998

Protein kinase C disrupts cannabinoid actions by phosphorylation of the CB1 cannabinoid receptor.

We have found that phosphorylation of a G-protein-coupled receptor by protein kinase C (PKC) disrupts modulation of ion channels by the receptor. In AtT-20 cells transfected with rat cannabinoid receptor (CB1), the activation of an inwardly rectifying potassium current (Kir current) and depression of P/Q-type calcium channels by cannabinoids were prevented by stimulation of protein kinase C by 100 nM phorbol 12-myristate 13-acetate (PMA). In contrast, activation of Kir current by somatostatin was unaffected, and inhibition of calcium channels was only modestly attenuated. The possibility that PKC acted by phosphorylating CB1 receptors was confirmed by demonstrating that PKC phosphorylated a single serine (S317) of a fusion protein incorporating the third intracellular loop of CB1. Mutating this serine to alanine did not affect the ability of CB1 to modulate currents, but it eliminated disruption by PMA, demonstrating that PKC can disrupt ion channel modulation by receptor phosphorylation.

Analgesics↗
PubMed1998

Mitochondrial oversight of cellular Ca2+ signaling.

Mitochondria, the metabolic powerhouses of the cell, can sequester and release large amounts of Ca2+. This import and export of Ca2+ helps to adjust energy production to cellular needs. Recent advances show that mitochondrial Ca2+ fluxes play a major role in normal Ca2+ signaling.

Calcium↗
PubMed1997

Disruption of the m1 receptor gene ablates muscarinic receptor-dependent M current regulation and seizure activity in mice.

Muscarinic acetylcholine receptors are members of the G protein-coupled receptor superfamily expressed in neurons, cardiomyocytes, smooth muscle, and a variety of epithelia. Five subtypes of muscarinic acetylcholine receptors have been discovered by molecular cloning, but their pharmacological similarities and frequent colocalization make it difficult to assign functional roles for individual subtypes in specific neuronal responses. We have used gene targeting by homologous recombination in embryonic stem cells to produce mice lacking the m1 receptor. These mice show no obvious behavioral or histological defects, and the m2, m3, and m4 receptors continue to be expressed in brain with no evidence of compensatory induction. However, the robust suppression of the M-current potassium channel activity evoked by muscarinic agonists in sympathetic ganglion neurons is completely lost in m1 mutant mice. In addition, both homozygous and heterozygous mutant mice are highly resistant to the seizures produced by systemic administration of the muscarinic agonist pilocarpine. Thus, the m1 receptor subtype mediates M current modulation in sympathetic neurons and induction of seizure activity in the pilocarpine model of epilepsy.

Animals↗