• The Korean Journal of Physiology and Pharmacology
• /
• v.11 no.5
• /
• pp.197-206
• /
• 2007

The aim of the present study was to investigate the effects of 6-chloro-7,8-dihydroxy-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine(SKF81297), a selective agonist of dopaminergic $D_1$ receptor, on the secretion of catecholamines(CA) evoked by cholinergic stimulation and membrane-depolarization in the isolated perfused rat adrenal gland, and also to elucidate the mechanism involved. SKF81297($10{\sim}100{\mu}M$) perfused into an adrenal vein for 60 min produced dose- and time-dependent inhibition of CA secretory responses evoked by ACh(5.32 mM), high $K^+$(56 mM), DMPP($100{\mu}M$) and McN-A-343($100{\mu}M$). Also, in adrenal glands loaded with SKF81297($30{\mu}M$), the CA secretory responses evoked by Bay-K-8644($10{\mu}M$), an activator of L-type $Ca^{2+}$ channels and cyclopiazonic acid($10{\mu}M$), an inhibitor of cytoplasmic $Ca^{2+}$-ATPase were also inhibited. However, in the presence of the dopamine $D_1$ receptor antagonist, (R)-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-benzazepine-7-ol(SCH23390, $3{\mu}M$), which is a selective antagonist of dopaminergic $D_1$ receptor, the inhibitory responses of SKF81297($30{\mu}M$) on the CA secretion evoked by ACh, high $K^+$, DMPP, McN-A-343, Bay-K-8644, and cyclopiazonic acid were significantly reduced. Collectively, these experimental results suggest that SKF81297 inhibits the CA secretion from the rat adrenal medulla evoked by cholinergic stimulation(both nicotininc and muscarinic receptors) and membrane depolarization. This inhibitory of SKF81297 seems to be mediated by stimulation of dopaminergic $D_1$ receptors located on the rat adrenomedullary chromaffin cells, which are relevant to extra- and intracellular calcium mobilization. Therefore, it is thought that the presence of the dopaminergic $D_1$ receptors may be involved in regulation of CA release in the rat adrenal medulla.

• The Korean Journal of Pharmacology
• /
• v.27 no.1
• /
• pp.45-52
• /
• 1991

The inhibitory effects of GS354 and GS389 on cytosolic $Ca^{2+}$ level ($[Ca^{2+}]_{1}$; measured with fura-2 fluorescence) and muscle tension in vascular smooth muscle of rat thoracic aorta were investigated. Both GS354 and GS389 inhibited the contractions induced by high $K^+$ or by norepinephrine. The vasodilator effect of GS354 was accompanied by a decrease in $[Ca^{2+}]_{1}$. The inhibitory effect on high $K^+-stimulated$ $[Ca^{2+}]_{1}$ was antagonized by a $Ca^{2+}$ channel activator, Bay K8644. However, the inhibitory effect on muscle tension was not antagonized by Bay K8644. These results suggest that GS354 inhibits $Ca^{2+}$ channels to decrease $[Ca^{2+}]_{1}$ and also decreases $Ca^{2+}$ sensitivity of contractile elements. The inhibitory effects of GS389 was accompanied by the increase in tissue fluorescence. This increment was not due to fura-2 fluorescence but to endogeneous pyridine nucleotides, suggesting that GS389 has an effect to inhibit mitochondrial function. Because of this interference, effects of GS389 on $[Ca^{2+}]_{1}$ was obscured. However, since sequential addition of Bay K8644 in the presence of GS389 further increased the fluorescence but not muscle tension, this compound seems to have the effects to inhibit $Ca^{2+}$ channels and to decrease $Ca^{2+}$ sensitivity of contractile elements.

• International Journal of Oral Biology
• /
• v.45 no.2
• /
• pp.58-63
• /
• 2020

The salivary glands secrete saliva, which plays a role in the maintenance of a healthy oral environment. Under physiological conditions, saliva secretion within the acinar cells of the gland is regulated by stimulation of specific calcium (Ca²⁺) signaling mechanisms such as increases in the intracellular Ca²⁺ concentration ([Ca²⁺]_i) via storeoperated Ca²⁺ entry, which involves components such as Orai1, transient receptor potential (TRP) canonical 1, stromal interaction molecules, and inositol 1,4,5-triphosphate (IP₃) receptors (IP₃Rs). Homer proteins are scaffold proteins that bind to G protein-coupled receptors, IP₃Rs, ryanodine receptors, and TRP channels. However, their exact role in Ca²⁺ signaling in the salivary glands remains unknown. In the present study, we investigated the role of Homer2 in Ca²⁺ signaling and saliva secretion in parotid gland acinar cells under physiological conditions. Deletion of Homer2 (Homer2^-/-) markedly decreased the amplitude of [Ca²⁺]_i oscillations via the stimulation of carbachol, which is physiologically concentrated in parotid acinar cells, whereas the frequency of [Ca²⁺]_i oscillations showed no difference between wild-type and Homer2^-/- mice. Homer2^-/- mice also showed a significant decrease in amylase release by carbachol in the parotid gland in a dose-dependent manner. These results suggest that Homer2 plays a critical role in maintaining [Ca²⁺]_i concentration and secretion of saliva in mouse parotid gland acinar cells.

• International Journal of Oral Biology
• /
• v.34 no.2
• /
• pp.73-79
• /
• 2009

Cytosolic $Ca^{2+}$ is an important regulator of tumor cell proliferation and metastasis. Recently, the strategy of blocking receptors and channels specific to certain cancer cell types has emerged as a potentially viable future treatment. Oral squamous cell carcinoma is an aggressive form of cancer with a high metastasis rate but the receptor-mechanisms involved in $Ca^{2+}$ signaling in these tumors have not yet been elucidated. In our present study, we report that bradykinin induces $Ca^{2+}$ signaling and its modulation in the human oral squamous carcinoma cell line, HSC-3. Bradykinin was found to increase the cytosolic $Ca^{2+}$ levels in a concentration-dependent manner. This increase was inhibited by pretreatment with the phospholipase C-${\beta}$ inhibitor, U73122, and also by 2-aminoethoxydiphenyl borate, an inhibitor of the inositol 1,4,5-trisphosphate receptor. Pretreatment with extracellular ATP also inhibited the peak bradykinin-induced $Ca^{2+}$ rise. In contrast, the ATP-induced rise in cytosolic $Ca^{2+}$ was not affected by pretreatment with bradykinin. Pretreatment of the cells with either forskolin or phorbol 12-myristate 13-acetate (activators of adenylyl cyclase and protein kinase C, respectively) prior to bradykinin application accelerated the recovery of cytosolic $Ca^{2+}$ to baseline levels. These data suggest that bradykinin receptors are functional in $Ca^{2+}$ signaling in HSC-3 cells and may therefore represent a future target in treatment strategies for human oral squamous cell carcinoma.

• Biomolecules & Therapeutics
• /
• v.21 no.2
• /
• pp.121-125
• /
• 2013

Hesperetin (3',5,7-trihydroxy 4'-methoxyflavanone) and its glycoside hesperidin (hesperetin 7-rhamnoglucoside) in oranges have been reported to possess pharmacological effects related to anti-obesity. However, hesperetin and hesperidin have not been studied on suppressive effects on appetite. This study examined that hesperetin and hesperidin can stimulate the release of cholecystokinin (CCK), one of appetite-regulating hormones, from the enteroendocrine STC-1 cells, and then examined the mechanisms involved in the CCK release. Hesperetin significantly and dose-dependently stimulated CCK secretion with an $EC_{50}$ of 0.050 mM and increased the intracellular $Ca^{2+}$ concentrations ($[Ca^{2+}]_i$) compared to the untreated control. The stimulatory effect by hesperetin was mediated via the entry of extracellular $Ca^{2+}$ and the activation of TRP channels including TRPA1. These results suggest that hesperetin can be a candidate biomolecule for the suppression of appetite and eventually for the therapeutics of obesity.

• The Korean Journal of Physiology
• /
• v.26 no.2
• /
• pp.113-122
• /
• 1992

It is well known that chromaffin cells of adrenal medulla secrete catecholamine in response to sympathetic nerve activation and the influx of $Ca^{2+}$ through the voltage dependent $Ca^{2+}$ channels (VDCC) in the cell membrane do a major role in this secretory process. In this study, we explored the effect of divalent cations on VDCC of rat chromaffin cells. Rat (Sprague-Dawley rat, 150-250 gm) chromaffin cells were isolated and cultured. Standard giga seal, whole cell recording techniques were employed to study $Ca^{2+}$ current with external and internal solutions that could effectively isolate VDCC currents $(NMG\;in\;external\;and\;TEA\;and\;Cs^{2+}\;in\;internal\;solution)$. The voltage dependence and the inactivation time course of VDCC in our cells were identical to those of bovine chromaffin cells. A persistent inward current was first activated by depolarizing step pulse from the holding potential (H.P.) of -80 mV to -40 mV, increased to maximum amplitude at around +10 mV, and became smaller with progressively higher depolarizing pulses to reverse at around +60 mV. The inactivation time constant $(\tau)$, fitted from the long duration test potential (2 sec) was $1295.2{\pm}126.8$ msec $(n=20,\;1\;day\;of\;culture,\;mean\;{\pm}S.E.M.)$ and the kinetic parameters were not altered along the culture duration. Nicardipine $(10\;{\mu}M)$ blocked the current almost completely. Among treated divalent cations such as $Cd^{2+},\;Co^{2+},\;Ni^{2+},\;Zn^{2+}\;and\;,Mn^{2+},\;Cd^{2+}$ was the most potent blocker on VDCC. When the depolarizing step pulse from -80 mV to 10 mV was applied, the equilibrium dissociation constant $(K_d)$ of $Cd^{2+}\;was\;39\;{\mu}M,\;K_d\;of\;Co^{2+}\;was\;100\;{\mu}M\;and\;K_d\;of\;Ni^{2+}];was];780{\mu}M.$ The principal findings of this study are as follows. First, the majority of $Ca^{2+}$ channels in rat chromaffin cells are well classified to L-type $Ca^{2+}$ channel in the view of kinetics and pharmacology. Second, all divalent cations tested could block the $Ca^{2+}$ current and the most potent blocker among the tested was $Cd^{2+}$.

• The Korean Journal of Physiology and Pharmacology
• /
• v.10 no.2
• /
• pp.59-64
• /
• 2006

The effects of $Zn^{2+}$ on spontaneous glutamate and GABA release were tested in mechanically dissociated rat CA3 pyramidal neurons which retained functional presynaptic nerve terminals. The spontaneous miniature excitatory and inhibitory postsynaptic currents (mEPSCs and mIPSCs, respectively) were pharmacologically isolated and recorded using whole-cell patch clamp technique under voltage-clamp conditions. $Zn^{2+}$ at a lower concentration $(30{\mu}M)$ increased GABAergic mIPSC frequency without affecting mIPSC amplitude, but it decreased both mIPSC frequency and amplitude at higher concentrations $({\ge}300{\mu}M)$. In contrast, $Zn^{2+}$ (3 to $100{\mu}M$) did not affect glutamatergic mEPSCs, although it slightly decreased both mIPSC frequency and amplitude at $300{\mu}M$ concentration. Facilitatory effect of $Zn^{2+}$ on GABAergic mIPSC frequency was occluded either in $Ca^{2+}$-free external solution or in the presence of $100{\mu}M$ 4-aminopyridine, a non-selective $K^{+}$ channel blocker. The results suggest that $Zn^{2+}$ at lower concentrations depolarizes GABAergic nerve terminals by blocking $K^{+}$ channels and increases the probability of spontaneous GABA release. This $Zn^{2+}$-mediated modulation of spontaneous GABAergic transmission is likely to play an important role in the regulation of neuronal excitability within the hippocampal CA3 area.

• The Korean Journal of Physiology and Pharmacology
• /
• v.7 no.4
• /
• pp.223-229
• /
• 2003

Using phospholipase D1 (PLD1)-overexpressing PC12 (PLD1-PC12) cells, the regulatory roles of PLD1 on ATP-induced currents were investigated. In control and PLD1-PC12 cells, ATP increased PLD activity in an external $Ca^{2+}$ dependent manner. PLD activity stimulated by ATP was substantially larger in PLD1-PC12 cells than in control cells. In whole-cell voltage-clamp mode, ATP induced transient inward and outward currents. The outward currents inhibited by TEA or charybdotoxin were significantly larger in PLD1-PC12 cells than in control cells. The inward currents known as $Ca^{2+}$ permeable nonselective cation currents were also larger in PLD1-PC12 cells than in control cells. However, the difference between the two groups of cells disappeared in $Ca^{2+}$-free external solution, where ATP did not activate PLD. Finally, ATP-induced $^{45}Ca$ uptakes were also larger in PLD1-PC12 cells than in control cells. These results suggest that PLD enhances ATP-induced $Ca^{2+}$ influx via $Ca^{2+}$ permeable nonselective cation channels and increases subsequent $Ca^{2+}$-activated $K^+$ currents in PC12 cells.

• Molecules and Cells
• /
• v.43 no.1
• /
• pp.66-75
• /
• 2020

Saturated fatty acids contribute to β-cell dysfunction in the onset of type 2 diabetes mellitus. Cellular responses to lipotoxicity include oxidative stress, endoplasmic reticulum (ER) stress, and blockage of autophagy. Palmitate induces ER Ca²⁺ depletion followed by notable store-operated Ca²⁺ entry. Subsequent elevation of cytosolic Ca²⁺ can activate undesirable signaling pathways culminating in cell death. Mitochondrial Ca²⁺ uniporter (MCU) is the major route for Ca²⁺ uptake into the matrix and couples metabolism with insulin secretion. However, it has been unclear whether mitochondrial Ca²⁺ uptake plays a protective role or contributes to lipotoxicity. Here, we observed palmitate upregulated MCU protein expression in a mouse clonal β-cell, MIN6, under normal glucose, but not high glucose medium. Palmitate elevated baseline cytosolic Ca²⁺ concentration ([Ca²⁺]_i) and reduced depolarization-triggered Ca²⁺ influx likely due to the inactivation of voltage-gated Ca²⁺ channels (VGCCs). Targeted reduction of MCU expression using RNA interference abolished mitochondrial superoxide production but exacerbated palmitate-induced [Ca²⁺]_i overload. Consequently, MCU knockdown aggravated blockage of autophagic degradation. In contrast, co-treatment with verapamil, a VGCC inhibitor, prevented palmitate-induced basal [Ca²⁺]_i elevation and defective [Ca²⁺]_i transients. Extracellular Ca²⁺ chelation as well as VGCC inhibitors effectively rescued autophagy defects and cytotoxicity. These observations suggest enhanced mitochondrial Ca²⁺ uptake via MCU upregulation is a mechanism by which pancreatic β-cells are able to alleviate cytosolic Ca²⁺ overload and its detrimental consequences.

• The Korean Journal of Physiology and Pharmacology
• /
• v.15 no.1
• /
• pp.53-59
• /
• 2011

The secretion of insulin from pancreatic ${\beta}$-cells is triggered by the influx of $Ca^{2+}$ through voltage-dependent $Ca^{2+}$ channels. The resulting elevation of intracellular calcium ($[Ca^{2+}]_i$) triggers additional $Ca^{2+}$ release from internal stores. Less well understood are the mechanisms involved in $Ca^{2+}$ mobilization from internal stores after activation of $Ca^{2+}$ influx. The mobilization process is known as calcium-induced calcium release (CICR). In this study, our goal was to investigate the existence of and the role of caffeine-sensitive ryanodine receptors (RyRs) in a rat pancreatic ${\beta}$-cell line, INS-1 cells. To measure cytosolic and stored $Ca^{2+}$, respectively, cultured INS-1 cells were loaded with fura-2/AM or furaptra/AM. $[Ca^{2+}]_i$ was repetitively increased by caffeine stimulation in normal $Ca^{2+}$ buffer. However, peak $[Ca^{2+}]_i$ was only observed after the first caffeine stimulation in $Ca^{2+}$ free buffer and this increase was markedly blocked by ruthenium red, a RyR blocker. KCl-induced elevations in $[Ca^{2+}]_i$ were reduced by pretreatment with ruthenium red, as well as by depletion of internal $Ca^{2+}$ stores using cyclopiazonic acid (CPA) or caffeine. Caffeine-induced $Ca^{2+}$ mobilization ceased after the internal stores were depleted by carbamylcholine (CCh) or CPA. In permeabilized INS-1 cells,$Ca^{2+}$ release from internal stores was activated by caffeine, $Ca^{2+}$, or ryanodine. Furthermore, ruthenium red completely blocked the CICR response in perrneabilized cells. RyRs were widely distributed throughout the intracellular compartment of INS-1 cells. These results suggest that caffeine-sensitive RyRs exist and modulate the CICR response from internal stores in INS-1 pancreatic ${\beta}$-cells.