Phosphate regulates osteopontin gene transcription.

Extracellular inorganic phosphate (ePi) is a key regulator of cementoblast behavior, both in vivo and in vitro, and results in a marked increase in osteopontin expression in vitro. To examine the molecular mechanisms involved in ePi induction of osteopontin gene expression, we transfected a series of osteopontin promoter-luciferase constructs into OCCM-30 cementoblasts. Our results demonstrate that ePi can directly induce osteopontin gene transcription. The region responsive to ePi signaling was localized to a 53-bp region of the promoter between -1454 and -1401 that contains a glucocorticoid response element (GRE). Mutation of the GRE abolished the ePi response, suggesting that glucocorticoid receptor (GR) signaling is required for ePi-mediated transcription. In addition, treatment of cells with the GR antagonist RU-486 (Mifepristone) prevented promoter activation by ePi. The results presented support a model demonstrating that inorganic phosphate regulates OPN gene transcription in cementoblasts through a pathway that requires a functional GR.

Bone formation by BMP-7-transduced human gingival keratinocytes.

BMPs are a family of pleiotropic signaling molecules involved at various stages in the formation of bones and teeth. In addition, recombinant BMP can induce bone and dentin regeneration when applied directly to adult tissues. We have shown that fibroblasts transduced ex vivo by BMP cDNA delivered by recombinant adenoviruses induce bone formation and convert to osteoblasts upon implantation in vivo. To determine if this osteogenic capacity was limited to fibroblasts, we found that BMP-7-transduced human oral keratinocyte cells (HOKC) also formed ectopic bone. The ossicles formed by the BMP-7-transduced HOKC were smaller and more dense than those formed by BMP-7-transduced human gingival fibroblasts (HGF). Implanted HOKC were localized adjacent to the developing bone by immunocytochemical detection of keratin expression. However, no bone-like tissue formed when HOKC were implanted into diffusion chambers in vivo. We conclude that BMP-transduced HOKC secrete BMP and form bone in vivo but, unlike BMP-transduced HGF, do not transdifferentiate to osteoblasts.

Involvement of calcium in interactions between gingival epithelial cells and Porphyromonas gingivalis.

Porphyromonas gingivalis, a periodontal pathogen can invade primary cultures of gingival epithelial cells. This invasion was significantly inhibited (74-81%) by thapsigargin and 1,2-bis(2-aminophenoxy)ethane-N,N,N1,N1-tetraacetic acid, acetoxymethyl ester (BAPTA/AM), but not by EDTA or amiloride. Release of Ca2+ from an intracellular store and the subsequent increase in cytosolic [Ca2+] may, therefore, be involved in the invasion process, while Ca2+ influx is not. Moreover, cytosolic [Ca2+] was found to increase transiently in about 30% of gingival epithelial cells acutely exposed to P. gingivalis, but not in unexposed cells, or in cells exposed to noninvasive Escherichia coli. These findings indicate that P. gingivalis invasion of epithelial cells is correlated with activation of [Ca2+]-dependent host cell signaling systems.

Absence of acetylcholine- and ionomycin-activated Cl- currents in submandibular cells of early postnatal rats.

Using the whole-cell patch-clamp technique, we investigated developmental changes in the expression of an acetylcholine- (Ach-) activated Cl- conductance in rat submandibular acinar cells. ACh induced an oscillatory inward current in cells isolated from animals older than 5 weeks, but not in animals less than 2-3 weeks of age. The current/voltage (I/V) relationship of the ACh-induced current was that of an outward rectifier, and the current was inhibited by intracellular BAPTA, a Ca2+ buffer, indicating the current was Ca2+ activated. The ACh-induced current was also blocked in the presence of DPC and SITS, two Cl- current inhibitors in other tissues. Ionomycin mimicked the effect of ACh but in a nonoscillatory fashion. The appearance of the ionomycin-induced currents was also age related, as the current was not observed to occur in animals less than 2-3 weeks old. Since both ACh and ionomycin significantly increase cytosolic [Ca2+] in the acinar cells of young animals, the correlation between the age dependence of the ACh-activated Cl- current and the ionomycin-activated Cl- current responses suggests that the lack of responsiveness observed in the young animals is due to the absence of Ca2+-activated Cl- channels, rather than to a deficiency of a cellular mediator.

Mycoplasma orale infection affects K+ and Cl- currents in the HSG salivary gland cell line.

The relations between K+ channel and Cl- channel currents and mycoplasma infection status were studied longitudinally in HSG cells, a human submandibular gland cell line. The K+ channel currents were disrupted by the occurrence of mycoplasma infection: muscarinic activation of K+ channels and K+ channel expression as estimated by ionomycin- or hypotonically induced K+ current responses were all decreased. Similar decreases in ionomycin- and hypotonically induced responses were observed for Cl- channels, but only the latter decrease was statistically significant. Also, Cl- currents could be elicited more frequently than K+ currents (63% of cases versus 0%) in infected cells when tested by exposure to hypotonic media, indicating that mycoplasma infection affects K+ channels relatively more than Cl- channels. These changes occurred in the originally infected cells, were ameliorated when the infection was cleared with sparfloxacin, and recurred when the cells were reinfected. Such changes would be expected to result in hyposecretion of salivary fluid if they occurred in vivo.

Hypotonically activated chloride current in HSG cells.

Hypotonically induced changes in whole-cell currents and in cell volume were studied in the HSG cloned cell line using the whole-cell, patch clamp and Coulter counter techniques, respectively. Exposures to 10 to 50% hypotonic solutions induced dose-dependent increases in whole-cell conductances when measured using K+ and Cl- containing solutions. An outward current detected at 0 mV, corresponded to a K+ current which was transiently activated, (usually preceding activation of an inward current and had several characteristics in common with a Ca(2+)-activated K+ current we previously described in these cells. The hypotonically induced inward current had characteristics of a Cl- current. This current was inhibited by NPPB (5-nitro-2-(3-phenyl-propylamino)-benzoate) and SITS (4-acetamido-4'-isothiocyanostilbene), and its reversal potentials corresponded to the Cl- equilibrium potentials at high and low external Cl- concentrations. The induced current inactivated at voltages greater than +80 mV, and the I-V curve was outwardly rectifying. The current was unaffected by addition of BAPTA or removal of GTP from the patch pipette, but was inhibited by removal of ATP or by the presence of extracellular arachidonic acid, quinacrine, nordihydroguairetic acid, and cytochalasin D. Moreover, exposure of HSG cells to hypotonic media caused them to swell and then to undergo a regulatory volume decrease (RVD) response. Neither NPPB, SITS or quinine acting alone could inhibit RVD, but NPPB and quinine together totally inhibited RVD. These properties, plus the magnitudes of the induced currents, indicate that the hypotonically induced K+ and Cl- currents may underlie the RVD response. Cytochalasin D also blocked the RVD response, indicating that intact cytoskeletal F-actin may be required for activation of the present currents. Hence, our results indicate that hypotonic stress activates K+ and Cl- conductances in these cells, and that the activation pathway for the K+ conductance apparently involves [Ca2+], while the activation pathway for the Cl- conductance does not involve [Ca2+] nor lipoxygenase metabolism, but does require intact cytoskeletal F-actin.

Characteristics and regulation of a muscarinically activated K current in HSG-PA cells.

Whole cell currents were measured in HSG-PA cells (a proposed model for salivary gland duct cells) after muscarinic receptor activation or exposure to known signaling agents. Exposure to carbachol or oxotremorine M produced large and often oscillatory increases in outward current whose reversal potentials indicated a K current. The current was sensitive to extracellular atropine, charybdotoxin, and quinine, but not apamin, and to 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid in the pipette. The response was prolonged or increased by guanosine 5'-O-(3-thiotriphosphate) and mimicked by D-myo-inositol 1,4,5-trisphosphate (IP3) or heparin in the pipette and by extracellular Ca ionophores. Tetraethylammonium indirectly inhibited the response via the muscarinic receptor. Fura 2 in cell suspensions showed that muscarinic agonists increased cytosolic Ca ion concentration ([Ca2+]i) five- to sevenfold, and measurements with indo 1 in individual cells showed that the oscillatory changes in outward current were tightly correlated with parallel changes in [Ca2+]i. The results indicate that muscarinic receptor stimulation of HSG-PA cells activates Ca(2+)-activated K channels through a signaling pathway involving a G protein, IP3 production, and increased [Ca2+]i levels. These findings are similar to those in salivary gland acinar cells.

Two sites for adenine-nucleotide regulation of ATP-sensitive potassium channels in mouse pancreatic beta-cells and HIT cells.

ATP-inhibited potassium channels (K(ATP)) were studied in excised, inside-out patches from cultured adult mouse pancreatic beta-cells and HIT cells. In the absence of ATP, ADP opened K(ATP) channels at concentrations as low as 10 microM and as high as 500 microM, with maximal activation between 10 and 100 microM ADP in mouse beta-cell membrane patches. At concentrations greater than 500 microM, ADP inhibited K(ATP) channels while 10 mM virtually abolished channel activity. HIT cell channels had a similar biphasic response to ADP except that more than 1 mM ADP was required for inhibition. The channel opening effect of ADP required magnesium while channel inhibition did not. Using creatine/creatine phosphate solutions with creatine phosphokinase to fix ATP and ADP concentrations, we found substantially different K(ATP)-channel activity with solutions having the same ATP/ADP ratio but different absolute total nucleotide levels. To account for ATP-ADP competition, we propose a new model of channel-nucleotide interactions with two kinds of ADP binding sites regulating the channel. One site specifically binds MgADP and increases channel opening. The other, the previously described ATP site, binds either ATP or ADP and decreases channel opening. This model very closely fits the ADP concentration-response curve and, when incorporated into a model of beta-cell membrane potential, increasing ADP in the 10 and 100 microM range is predicted to compete very effectively with millimolar levels of ATP to hyperpolarize beta-cells. The results suggest that (i) K(ATP)-channel activity is not well predicted by the "ATP/ADP ratio," and (ii) ADP is a plausible regulator of K(ATP) channels even if its free cytoplasmic concentration is in the 10-100 microM range as suggested by biochemical studies.

Specificity of tetraethylammonium and quinine for three K channels in insulin-secreting cells.

The effects of tetraethylammonium (TEA) and quinine on Ca-activated [K(Ca)], ATP-sensitive [K(ATP)]K channels and delayed-rectifier K current [K(dr)] have been studied in cultured insulin-secreting HIT cells using the patch-clamp technique. K(Ca) and K(ATP) channels were identified in excised, outside/out patches using physiological solutions and had unitary conductances of 60.8 +/- 1.3 pS (n = 31) and 15.4 +/- 0.3 pS (n = 40), respectively. Macroscopic K(dr) current (peak current = 607 +/- 100 pA at +50 mV, n = 14) were recorded in the presence of 100 microM cadmium and 0.5 microM tetrodotoxin. Tetraethylammonium (TEA) blocked all three channel types but was more effective on K(Ca) channels (EC50 = 0.15 mM) than on K(ATP) channels (EC50 = 15 mM) or K(dr) currents (EC50 = 3 mM). Quinine also blocked all three currents but was less effective on K(Ca) channels (EC50 = 0.3 mM) while equally effective against K(ATP) channels and K(dr) currents (EC50 = 0.025 mM). TEA blocked K(Ca) and K(ATP) channels by reducing their single-channel conductances and decreasing the probability of K(ATP) channel opening. Quinine blocked K(Ca) channels by reducing the single-channel conductance, but blocked K(ATP) channels by reducing the probability of channel opening. Reinterpretation of previous microelectrode studies in light of these findings suggest that, (i) only K(ATP) channels are active in low glucose, (ii) both K(Ca) and K(dr) channels may assist Ca-spike repolarization, and (iii) K(Ca) channels play no role in forming the burst pattern of Ca spiking in the B cell.

The oral hypoglycemic agent, U-56324, inhibits the activity of ATP-sensitive potassium channels in cell-free membrane patches from cultured mouse pancreatic B-cells.

U-56324, a hypoglycemic agent derived from nicotinic acid, inhibited the activity of ATP-sensitive potassium channels in excised patches from mouse pancreatic B-cells. The effect of U-56324 on channel activity was reversible and concentration-dependent while it had no effect on single channel conductance. The positional isomer, U-59588, which has relatively little hypoglycemic activity, had no effect on channel properties. U-56324, at the same concentrations, had no effect on calcium-activated potassium channels. The basis for the potentially antidiabetic properties of U-56324 may therefore be due to direct and specific inhibition of ATP-sensitive potassium channels.

Neuropeptide Y mimics a non-adrenergic component of sympathetic vasoconstriction in the bullfrog.

The effects of preganglionic sympathetic nerve stimulation and exogenous agents upon vascular tone were observed in hindlimb preparations of pithed adult bullfrogs. Repetitive electrical stimulation of the sympathetic C, but not the B, system elicited arterial vasoconstriction and reduced blood flow in vascular beds supplying the sartorius muscle and the skin. Close-arterial injections of epinephrine and neuropeptide Y each mimicked neurogenic vasoconstriction. After close-arterial injection of phentolamine, an alpha-adrenergic antagonist, the maximal effects of nerve stimulation were delayed in onset and reduced in magnitude, but not eliminated. Pretreatment with phentolamine blocked the vasoconstriction caused by injection of epinephrine, and produced a mild reduction in responses to neuropeptide Y. These observations demonstrate the vasomotor function of the sympathetic C system and they support the hypothesis that neuropeptide Y and epinephrine function as cotransmitters in postganglionic C neurons.

Expression of a rapid, low-voltage threshold K current in insulin-secreting cells is dependent on intracellular calcium buffering.

Depolarization-activated outward currents ranging in amplitude from 100-1000 pA were studied in cultured, insulin-secreting HIT cells and mouse B-cells using the whole-cell patch clamp. Outward current was identified as a K current since it was blocked by K channel blockers and its tail current reversed near EK. The K currents of HIT cells dialyzed with internal solutions containing 0.1-10 mM EGTA with no added calcium (Ca), or 10 mM EGTA with 2 mM added Ca, activated rapidly with depolarization. However, the stronger Ca buffer BAPTA (5 mM; no added Ca) blocked the rapidly activating current to reveal an underlying more slowly activating K current. With intracellular EGTA, application of the Ca channel blocker cadmium mimicked the effect of intracellular BAPTA. These data suggest that the rapid K current was mediated by low-voltage threshold, Ca-activated K channels while the slower K current was mediated by high threshold delayed rectifier K channels. Mouse B-cells also had both K current components. Dialyzing these cells with either BAPTA (5 mM, no added Ca) or high EGTA (10 mM with 2 mM Ca) blocked the rapid Ca-activated K current observed when cells were filled with 0.1 to 1 mM EGTA. It is concluded that the extent of Ca-activated K current activation in either HIT or adult mouse B-cells depends on the degree of intracellular Ca buffering.

Differential projections of B and C sympathetic axons in peripheral nerves of the bullfrog.

Accumulating evidence indicates that electrophysiologically distinct subsets of sympathetic neurons selectively innervate different classes of targets. The organization of this system may therefore be reflected in the sympathetic fiber contents of peripheral nerves. To test this possibility, we have mapped the pathways followed by three groups of postganglionic sympathetic axons in the bullfrog by recording compound action potentials and by retrograde tracing with horseradish peroxidase (HRP). The axons that were studied arise from fast B, slow B, and C-type neurons in ganglia 9 and 10 at the lumbar end of the paravertebral sympathetic chain. They project to peripheral targets primarily by way of the sciatic nerve and can be distinguished by the velocities with which they conduct action potentials. Action potentials were recorded with suction electrodes from isolated preparations composed of paravertebral chain ganglia 7-10, the sciatic nerve, and branches of the sciatic nerve that supply striated muscles, skin, and the bladder. Preganglionic B fibers were selectively activated by stimulating the paravertebral chain rostral to ganglion 7, and preganglionic C fibers were selectively activated by stimulating spinal nerves 7 and 8 at points central to their rami communicantes. Compound action potentials recorded from the sciatic, peroneal, tibial, and sural nerves and from the primary trunk of the pelvic nerve were each found to contain three components produced, respectively, by fast B, slow B, and C-type sympathetic axons. Similarly, action potentials recorded from cutaneous branches of the sciatic tree were found to contain three sympathetic components. By contrast, when compound action potentials were recorded from branches of the sciatic tree that directly enter and innervate striated muscles and also the bladder, the sympathetic responses were found to arise solely from C-type axons. HRP was used to label the sympathetic neurons that project to the sartorius muscle and into the cutaneous lateral crural nerve. Retrograde transport of HRP from the sartorius muscle labeled 17 +/- 4 (mean +/- s.d.) sympathetic neurons and 27 +/- 3 spinal motoneurons while transport from the lateral crural nerve labeled 68 +/- 47 sympathetic neurons but no spinal neurons. The average somatic diameter of ganglion cells projecting to the sartorius muscle was significantly smaller than that of cells projecting to the lateral crural nerve. The electrophysiological results indicate that fast B and slow B sympathetic axons in the sciatic trunk and its primary branches project selectively into cutaneous nerves while sympathetic C axons project into all peripheral nerves.(ABSTRACT TRUNCATED AT 400 WORDS)

Neuropeptide Y-like immunoreactivity in bullfrog sympathetic ganglia is restricted to C cells.

Staining the entire chain of paravertebral sympathetic ganglia in the bullfrog with an antiserum against porcine neuropeptide Y (NPY) revealed that, in each ganglion, a subpopulation of neurons expresses NPY-like immunoreactivity. Chromaffin cells in the sympathetic ganglia and in the adrenal gland were not stained by the anti-NPY serum. Since neurons in ganglia 9 and 10 of the sympathetic chain can be classified into 3 distinct groups on the basis of established electrophysiological criteria, we sought to identify the neurons in these ganglia that contain the NPY-like immunoreactivity. Accordingly, a series of cells was impaled with intracellular recording electrodes, identified as either fast B-, slow B-, or C-type neurons, filled with Lucifer yellow, and then processed with the anti-NPY serum for indirect immunofluorescence. These double-label experiments revealed that NPY-like immunoreactivity is expressed selectively by C-type sympathetic neurons. Counts of immunoreactive cells indicate that 55% of the neurons in ganglia 9 and 10 are C cells. A major target of the C cells appears to be the vasculature. In 3 different tissues innervated by sympathetic neurons, namely, the adrenal gland, the sartorius muscle, and the skin of the hindlimb, axons containing NPY-like immunoreactivity were found primarily within or near blood vessels. Within 14-60 d after extirpation of sympathetic ganglia 9 and 10, most immunoreactive axons in the sartorius muscle and nearby skin disappeared. In conjunction with other evidence, the possibility arises that sympathetic C cells function as vasomotor neurons that use an NPY-like substance and epinephrine as cotransmitters.

Intraseptal morphine potentiates pentobarbital narcosis and hypothermia in the rat.

Morphine injected intraseptally in the amounts of 35 and 70 nmol prolonged pentobarbital-induced narcosis in the rat. Pentobarbital-induced hypothermia was also potentiated by intraseptal injection of 70 nmol of morphine. These effects were antagonized when morphine was injected together with naltrexone (29 nmol). Naltrexone injected by itself into the septum did not significantly affect pentobarbital-narcosis and hypothermia. It is concluded that activation of mu opioid receptors in the septal region could affect the actions of pentobarbital.

Muscarinic receptors in rat sympathetic ganglia.

1 Potential changes in isolated superior cervical ganglia of the rat produced by muscarinic-receptor agonists were recorded by an extracellular ;air-gap' method.2 Muscarinic agonists produced a delayed low-amplitude ganglion depolarization, frequently preceded by a hyperpolarization. Potentials were enhanced by reducing [K(+)](o) or [Ca(2+)](o).3 Mean ED(50) values (muM) for depolarization at 25 degrees C were: oxotremorine 0.004, methylfurmethide 0.11, (+/-)-muscarine 0.24, furmethide 1.56, pilocarpine 4.81 and AHR-602 (N-benzylpyrrolidylacetate methobromide) 10.8. Responses produced by oxotremorine, pilocarpine and AHR-602 showed some characteristics of ;partial agonism'. ED(50) values (muM) for choline esters (measured in the presence of 2.5 mM hexamethonium) were: acetylcholine 3.2, methacholine 59 and bethanechol 78.4 Responses to muscarine were antagonized by hyoscine (K(I) 0.49 nM) atropine (K(I) 0.24 nM) methylscopolamine (K(I) 0.09 nM) lachesine (K(I) 0.15 nM) and (weakly) by hexamethonium (K(I) 0.2 mM). Propylbenzilylcholine mustard produced irreversible antagonism with an apparent onset rate constant of 2 x 10(5) M(-1)S(-1).5 Depolarization was accompanied by facilitation of submaximal ganglionic transmission.6 Muscarine (1 to 100 muM) initially reduced, then increased, the rate of (86)Rb(+)-efflux from isolated ganglia at both 6 and 120 mM [K(+)](o). These effects were reduced by 1 muM hyoscine.7 No consistent change in the amounts of cyclic 3',5'-guanosine monophosphate in isolated ganglia accompanying muscarinic depolarization could be detected.8 Mean against ED(50) values (muM) for contracting the rat isolated ileum were: oxotremorine 0.012, methylfurmethide 0.29, (+/-)-muscarine 0.48, pilocarpine 7.8 and AHR-602 9.9. Mean antagonist K(I) values (nM) were: hyoscine 0.17, atropine 0.34 and lachesine 0.27.9 It is concluded that ganglionic muscarinic receptors are quite similar to ileal receptors in terms of agonist ED(50) and antagonist K(I) values, and that the major difference between them lies in the greater ;efficacy' of certain agonists (pilocarpine, AHR-602 and McN-A-343) on the ganglion.