7submandibular glands from either wild-type or = 8 and 9 mice, respectively). from wild-type mice remained strongly hyperpolarized (?55 2 mV) relative to the Cl? equilibrium potential (?24 mV) during muscarinic stimulation. Similar hyperpolarizations were observed in and (but not level provides access to whole organ function, but the interpretation of results is complicated by systemic effects. In the latter case, saliva secretion is usually often induced by cholinergic agonists such as pilocarpine. It is well established that pilocarpine can stimulate saliva secretion through glandular muscarinic receptors. However, systemic administration of a cholinergic agonist also activates receptors in the brain and peripheral nervous system, stimulating saliva secretion through efferent innervation of the glands (Renzi 1993; Cecanho 1999; Renzi 2002; Takakura 2003). These systemic effects can be circumvented by studying surgically isolated salivary glands. Such assays give control over the composition of vascular perfusate including the agonist concentration. 1990; Ishikawa 1994), but this model has not been previously applied to mice. Here we implement and directly compare the and models in mice to confirm the details of the fluid secretion mechanism predicted at the single cell level. The currently accepted model for Cl?-dependent fluid secretion by acinar cells (reviewed by Cook 1994; Melvin 2005) postulates a key role for Ca2+-activated K+ channels for maintaining the membrane potential unfavorable to the Nernst potential for Cl?. Muscarinic stimulation initiates fluid secretion by activation of apical Ca2+-dependent Cl? channels, which mediate Cl? efflux into the acinar lumen. The associated charge movement produces a lumen-negative transepithelial potential that drives paracellular Na+ transport through tight junctions. The resulting luminal accumulation of NaCl generates an osmotic gradient necessary for transepithelial movement of water to produce a plasma-like primary secretion. Sustained secretion requires that this intracellular [Cl?] remains above its electrochemical equilibrium. Cytosolic Cl? is usually accumulated predominantly through a basolateral electroneutral Na+/K+/2Cl? cotransport mechanism, which is dependent around the inwardly directed Na+ gradient created by the Na+,K+-ATPase. Efflux of Cl? via apical channels depolarizes the cell membrane. Thus, it has been proposed that coordinated opening of basolateral Ca2+-activated K+ channels mediates outward K+ current to maintain the electrical driving force necessary for sustained Cl? secretion. Previous studies provided molecular and electrophysiological evidence for two types of Ca2+-activated K+ channels in parotid salivary gland acinar cells, the intermediate conductance (IK1) and large conductance (maxi-K) channels (Maruyama 1983; Wegman 1992; Park 2001; Nehrke 2003; Takahata 2003; Begenisich 2004; Romanenko 2006). The properties of IK1 channels are: (i) time- and voltage-independent currents, (ii) activation by submicromolar intracellular Ca2+ and by dcEBIO, (iii) inhibition by clotrimazole and TRAM-34, and (iv) 20C40 pS unitary conductance (Ishii 1997; Logsdon 1997; Jorgensen 1999; Begenisich 2004). The properties of maxi-K channels are also characteristically unique: (i) strong time dependence of the currents, (ii) strong outward rectification regulated by intracellular Ca2+, (iii) inhibition by paxilline, and (iv) large single-channel conductance of 100C300 pS (Latorre 1989; Pallanck & Ganetzky, 1994; Nehrke 2003; Salkoff 2006) (150C200 pS in mouse parotid acinar cells; Nehrke 2003; Thompson & Begenisich, 2006). Another unique feature of the maxi-K channel is usually its inhibition by IK1 current activation, which was exhibited in parotid acinar cells as well as in a heterologous expression system (Romanenko 2006; Thompson & Begenisich, 2006). The molecular identities of IK1 and maxi-K channels in parotid acinar cells were confirmed in mice in which the and genes, respectively, were disrupted (Begenisich 2004; Romanenko 2006). Surprisingly, loss of either IK1 or maxi-K channel expression did not impair parotid gland fluid secretion. However, disruptions of both the and genes produced a substantial (> 70%) reduction in secretion by mouse parotid glands (Romanenko 2006). Two Ca2+-activated K+ currents with properties similar to maxi-K channels are observed in mouse submandibular acinar cells (e.g. Maruyama 1983). IK1-like currents have also been characterized in rat submandibular acinar cells (Ishikawa 1994; Ishikawa & Murakami, 1995; Hayashi 2004), and maxi-K currents were found in human submandibular acinar cells (Morris 1987)..The resulting luminal accumulation of NaCl generates an osmotic gradient necessary for transepithelial movement of water to produce a plasma-like primary secretion. membrane potential in isolated submandibular acinar cells revealed mechanistic details underlying fluid secretion in K+ channel-deficient mice. The membrane potential of submandibular acinar cells from wild-type mice remained strongly hyperpolarized (?55 2 mV) relative to the Cl? equilibrium potential (?24 mV) during muscarinic stimulation. Similar hyperpolarizations were observed in and (but not level provides access to whole organ function, but the interpretation of results is complicated by systemic effects. In the latter case, saliva secretion is usually often induced by cholinergic agonists such as pilocarpine. It is well established that pilocarpine can stimulate saliva secretion through glandular muscarinic receptors. However, systemic administration of a cholinergic agonist also activates receptors in the brain and peripheral nervous system, stimulating saliva secretion through efferent innervation of the glands (Renzi 1993; Cecanho 1999; Renzi 2002; Takakura 2003). These systemic results could be circumvented by learning surgically isolated salivary glands. Such assays provide control over the structure of vascular perfusate like the agonist focus. 1990; Ishikawa 1994), but this model is not previously put on mice. Right here we put into action and directly evaluate the and versions in mice to verify the details from the liquid secretion mechanism expected at the solitary cell level. The presently approved model for Cl?-reliant liquid secretion by acinar cells (reviewed by Cook 1994; Melvin 2005) postulates an integral part for Ca2+-triggered K+ stations for keeping the membrane potential adverse towards the Nernst prospect of Cl?. Muscarinic excitement initiates liquid secretion by activation of apical Ca2+-reliant Cl? stations, which mediate Cl? efflux in to the acinar lumen. The connected charge motion generates a lumen-negative transepithelial potential that drives paracellular Na+ transportation through limited junctions. The ensuing luminal build up of NaCl generates an osmotic gradient essential for transepithelial motion of water to make a plasma-like major secretion. Continual secretion requires how the intracellular [Cl?] continues to be above its electrochemical equilibrium. Cytosolic Cl? can be accumulated mainly through a basolateral electroneutral Na+/K+/2Cl? cotransport system, which would depend for the inwardly aimed Na+ gradient developed from the Na+,K+-ATPase. Efflux of Cl? via apical stations depolarizes the cell membrane. Therefore, it’s been suggested that coordinated starting of basolateral Ca2+-triggered K+ stations mediates outward K+ current to keep up the electrical traveling force essential for suffered Cl? secretion. Earlier studies offered molecular and electrophysiological proof for just two types of Ca2+-triggered K+ stations in parotid salivary gland acinar cells, the intermediate conductance (IK1) and huge conductance (maxi-K) stations (Maruyama 1983; Wegman 1992; Recreation area 2001; Nehrke 2003; Takahata 2003; Begenisich 2004; Romanenko 2006). The properties of IK1 stations are: (i) period- and voltage-independent currents, (ii) activation by submicromolar intracellular Ca2+ and by dcEBIO, (iii) inhibition by clotrimazole and TRAM-34, and (iv) 20C40 pS unitary conductance (Ishii 1997; Logsdon 1997; Jorgensen 1999; Begenisich 2004). The properties of maxi-K stations will also be characteristically exclusive: (i) solid time dependence from the currents, (ii) solid outward rectification controlled by intracellular Ca2+, (iii) inhibition by paxilline, and (iv) huge single-channel conductance of 100C300 pS (Latorre 1989; Pallanck & Ganetzky, 1994; Nehrke 2003; Salkoff 2006) (150C200 pS in mouse parotid acinar cells; Nehrke 2003; Thompson & Begenisich, 2006). Another special feature from the maxi-K route can be its inhibition by IK1 current activation, that was proven in parotid acinar cells aswell as with a heterologous manifestation program (Romanenko 2006; Thompson & Begenisich, 2006). The molecular identities of IK1 and maxi-K stations in parotid acinar cells had been verified in mice where the and genes, respectively, had been disrupted (Begenisich 2004; Romanenko 2006). Remarkably, lack of either IK1 or maxi-K route expression didn’t impair parotid gland liquid secretion. Nevertheless, disruptions of Mouse monoclonal to E7 both and genes created a considerable (> 70%) decrease in secretion by mouse parotid glands (Romanenko 2006). Two Ca2+-triggered K+ currents with properties just like maxi-K stations are found in mouse submandibular acinar cells (e.g. Maruyama 1983). IK1-like.Glands were finely minced and digested for 5 min in Earle’s minimal necessary moderate (EMEM, Biofluid) containing 0.02% trypsin and 0.17 mg ml?1 Liberase RI Enzyme (Roche Applied Technology, Indianapolis, IN, USA), then centrifuged as well as the cell pellet rinsed in moderate containing trypsin inhibitor, accompanied by yet another 20 min of digestion in Liberase RI. with both K+ channel genes ablated C suggesting systemic complications using the assay strongly. Extra experiments concentrating on the membrane potential in isolated submandibular acinar cells exposed mechanistic details root liquid secretion in K+ channel-deficient mice. The membrane potential of submandibular acinar cells from wild-type mice continued to be highly hyperpolarized (?55 2 mV) in accordance with the Cl? equilibrium potential (?24 mV) during muscarinic excitement. Similar hyperpolarizations had been seen in and (however, not level provides usage of whole body organ function, however the interpretation of outcomes is challenging by systemic results. In the second option case, saliva secretion can be frequently induced by cholinergic agonists such as for example pilocarpine. It really is more developed that pilocarpine can promote saliva secretion through glandular muscarinic receptors. Nevertheless, systemic administration of the cholinergic agonist also activates receptors in the mind and peripheral anxious program, stimulating saliva secretion through efferent innervation from the glands (Renzi 1993; Cecanho 1999; Renzi 2002; Takakura 2003). These systemic results could be circumvented by learning surgically isolated salivary glands. Such assays provide control over the structure of vascular perfusate like the agonist focus. 1990; Ishikawa 1994), but this model is not previously put on mice. Right here we put into action and directly evaluate the and Methylene Blue versions in mice to verify the details from the liquid secretion mechanism forecasted at the one cell level. The presently recognized model for Cl?-reliant liquid secretion by acinar cells (reviewed by Cook 1994; Melvin 2005) postulates an integral function for Ca2+-turned on K+ stations for preserving the membrane potential detrimental towards the Nernst prospect of Cl?. Muscarinic arousal initiates liquid secretion by activation of apical Ca2+-reliant Cl? stations, which mediate Cl? efflux in to the acinar lumen. The linked charge motion creates a lumen-negative transepithelial potential that drives paracellular Na+ transportation through restricted junctions. The causing luminal deposition of NaCl generates an osmotic gradient essential for transepithelial motion of water to make a plasma-like principal secretion. Continual secretion requires which the intracellular [Cl?] continues to be above its electrochemical equilibrium. Cytosolic Cl? is normally accumulated mostly through a basolateral electroneutral Na+/K+/2Cl? cotransport system, which would depend over the inwardly aimed Na+ gradient made with the Na+,K+-ATPase. Efflux of Cl? via apical stations depolarizes the cell membrane. Hence, it’s been suggested that coordinated starting of basolateral Ca2+-turned on K+ stations mediates outward K+ current to keep the electrical generating force essential for suffered Cl? secretion. Prior studies supplied molecular and electrophysiological proof for just two types of Ca2+-turned on K+ stations in parotid salivary gland acinar cells, the intermediate conductance (IK1) and huge conductance (maxi-K) stations (Maruyama 1983; Wegman 1992; Recreation area 2001; Nehrke 2003; Takahata 2003; Begenisich 2004; Romanenko 2006). The properties of IK1 stations are: (i) period- and voltage-independent currents, (ii) activation by submicromolar intracellular Ca2+ and by dcEBIO, (iii) inhibition by clotrimazole and TRAM-34, and (iv) 20C40 pS unitary conductance (Ishii 1997; Logsdon 1997; Jorgensen 1999; Begenisich 2004). The properties of maxi-K stations may also be characteristically exclusive: (i) solid time dependence from the currents, (ii) solid outward rectification controlled by intracellular Ca2+, (iii) inhibition by paxilline, and (iv) huge single-channel conductance of 100C300 pS (Latorre 1989; Pallanck & Ganetzky, 1994; Nehrke 2003; Salkoff 2006) (150C200 pS in mouse parotid acinar cells; Nehrke 2003; Thompson & Begenisich, 2006). Another distinct feature from the maxi-K route is normally its inhibition by IK1 current activation, that was showed in parotid acinar cells aswell such as a heterologous appearance program (Romanenko 2006; Thompson & Begenisich, 2006). The molecular identities of IK1 and maxi-K stations in parotid acinar cells had been verified in mice in.= 5), that was not not the same as the existing in the wild-type mice ( significantly?14 4 pA pF?1; = 0.17). Liquid secretion in the assay was significantly (about 75%) low in pets with both K+ route genes ablated C highly suggesting systemic problems using the assay. Extra experiments concentrating on the membrane potential in isolated submandibular acinar cells uncovered mechanistic details root liquid secretion in K+ channel-deficient mice. The membrane potential of submandibular acinar cells from wild-type mice continued to be highly hyperpolarized (?55 2 mV) in accordance with the Cl? equilibrium potential (?24 mV) during muscarinic arousal. Similar hyperpolarizations had been seen in and (however, not level provides usage of whole body organ function, however the interpretation of outcomes is challenging by systemic results. In the last mentioned case, saliva secretion is normally frequently induced by cholinergic agonists such as for example pilocarpine. It really is more developed that pilocarpine can induce saliva secretion through glandular muscarinic receptors. Nevertheless, systemic administration of the cholinergic agonist also activates receptors in the mind and peripheral anxious program, stimulating saliva secretion through efferent innervation from the glands (Renzi 1993; Cecanho 1999; Renzi 2002; Takakura 2003). These systemic results could be circumvented by learning surgically isolated salivary glands. Such assays provide control over the structure of vascular perfusate like the agonist focus. 1990; Ishikawa 1994), but this model is not previously put on mice. Right here we put into action and directly evaluate the and versions in mice to verify the details from the liquid secretion mechanism forecasted at the one cell level. The presently recognized model for Cl?-reliant liquid secretion by acinar cells (reviewed by Cook 1994; Melvin 2005) postulates an integral function for Ca2+-turned on K+ stations for preserving the membrane potential detrimental towards the Nernst prospect of Cl?. Muscarinic arousal initiates liquid secretion by activation of apical Ca2+-reliant Cl? stations, which mediate Cl? efflux in to the acinar lumen. The linked charge motion creates a lumen-negative transepithelial potential that drives paracellular Na+ transportation through restricted junctions. The causing luminal deposition of NaCl generates an osmotic gradient essential for transepithelial motion of water to make a plasma-like principal secretion. Continual secretion requires which the intracellular [Cl?] continues to be above its electrochemical equilibrium. Cytosolic Cl? is normally accumulated mostly through a basolateral electroneutral Na+/K+/2Cl? cotransport system, which would depend over the inwardly aimed Na+ gradient made with the Na+,K+-ATPase. Efflux of Cl? via apical stations depolarizes the cell membrane. Hence, it’s been suggested that coordinated starting of basolateral Ca2+-turned on K+ stations mediates outward K+ current to keep the electrical generating force essential for suffered Cl? secretion. Prior studies supplied molecular and electrophysiological proof for just two types of Ca2+-turned on K+ stations in parotid salivary gland acinar cells, the intermediate conductance (IK1) and huge conductance (maxi-K) stations (Maruyama 1983; Wegman 1992; Recreation area 2001; Nehrke 2003; Takahata 2003; Begenisich 2004; Romanenko 2006). The properties of IK1 stations are: (i) period- and voltage-independent currents, (ii) activation by submicromolar intracellular Ca2+ and by dcEBIO, (iii) inhibition by clotrimazole and TRAM-34, and (iv) 20C40 pS unitary conductance (Ishii 1997; Logsdon 1997; Jorgensen 1999; Begenisich 2004). The properties of maxi-K stations may also be characteristically exclusive: (i) solid time dependence from the currents, (ii) solid outward rectification controlled by intracellular Ca2+, (iii) inhibition by paxilline, and (iv) huge single-channel conductance of 100C300 pS (Latorre 1989; Pallanck & Ganetzky, 1994; Nehrke 2003; Salkoff 2006) (150C200 pS in mouse parotid acinar cells; Nehrke 2003; Thompson & Begenisich, 2006). Another exclusive feature from the maxi-K route is certainly its inhibition by IK1 current activation, that was confirmed in parotid acinar cells aswell such as a heterologous appearance program (Romanenko 2006; Thompson & Begenisich, 2006). The molecular identities of IK1 and maxi-K stations in parotid acinar cells had been verified in mice where the and genes, respectively, had been disrupted (Begenisich 2004; Romanenko 2006). Amazingly, lack of either IK1 or maxi-K route expression didn’t impair parotid gland liquid secretion. Nevertheless, disruptions of both and genes created a considerable (> 70%) decrease in secretion by mouse parotid glands (Romanenko 2006). Two Ca2+-turned on K+ currents with properties comparable to maxi-K stations are found in mouse submandibular acinar cells (e.g. Maruyama 1983). IK1-like currents are also characterized in rat submandibular acinar cells (Ishikawa 1994; Ishikawa & Murakami, 1995; Hayashi 2004), and maxi-K currents had been found in individual submandibular acinar cells (Morris 1987). Right here we have motivated the physiological and pharmacological properties from the Ca2+-turned on IK1 and maxi-K stations in mouse submandibular acinar cells and confirmed their molecular identities using liquid secreted by perfused submandibular glands, however, not secretion, correlated with the electrophysiological measurements in.6). 2 mV) in accordance with the Cl? equilibrium potential (?24 mV) during muscarinic arousal. Similar hyperpolarizations had been seen in and (however, not level provides usage of whole body organ function, Methylene Blue however the interpretation of outcomes is challenging by systemic results. In the last mentioned case, saliva secretion is certainly frequently induced by cholinergic agonists such as for example pilocarpine. It really is more developed that pilocarpine can induce saliva secretion through glandular muscarinic receptors. Nevertheless, systemic administration of the cholinergic agonist also activates receptors in the mind and peripheral anxious program, stimulating saliva secretion through efferent innervation from the glands (Renzi 1993; Cecanho 1999; Renzi 2002; Takakura 2003). These systemic results could be circumvented by learning surgically isolated salivary glands. Such assays provide control over the structure of vascular perfusate like the agonist focus. 1990; Ishikawa 1994), but this model is not previously put on mice. Right here we put into action and directly evaluate the and versions in mice to verify the details from the liquid secretion mechanism forecasted at the one cell level. The presently recognized model for Cl?-reliant liquid secretion by acinar cells (reviewed by Cook 1994; Melvin 2005) postulates an integral function for Ca2+-turned on K+ stations for preserving the membrane potential harmful towards the Nernst prospect of Cl?. Muscarinic arousal initiates liquid secretion by activation of apical Ca2+-reliant Cl? stations, which mediate Cl? efflux in to the acinar lumen. The linked charge motion creates a lumen-negative transepithelial potential that drives paracellular Na+ transportation through restricted junctions. The causing luminal deposition of NaCl generates an osmotic gradient essential for transepithelial motion of water to make a plasma-like principal secretion. Continual secretion requires the fact that intracellular [Cl?] continues to be above its electrochemical equilibrium. Cytosolic Cl? is certainly accumulated mostly through a basolateral electroneutral Na+/K+/2Cl? cotransport system, which would depend in the inwardly aimed Na+ gradient made with the Na+,K+-ATPase. Efflux of Cl? via apical stations depolarizes the cell membrane. Thus, it has been proposed that coordinated opening of basolateral Ca2+-activated K+ channels mediates outward K+ current to maintain the electrical driving force necessary for sustained Cl? secretion. Previous studies provided molecular and electrophysiological evidence for two types of Ca2+-activated K+ channels in parotid salivary gland acinar cells, the intermediate conductance (IK1) and large conductance (maxi-K) channels (Maruyama 1983; Wegman 1992; Park 2001; Nehrke 2003; Takahata 2003; Begenisich 2004; Romanenko 2006). The properties of IK1 channels are: (i) time- and voltage-independent currents, (ii) activation by submicromolar intracellular Ca2+ and by dcEBIO, (iii) Methylene Blue inhibition by clotrimazole and TRAM-34, and (iv) 20C40 pS unitary conductance (Ishii 1997; Logsdon 1997; Jorgensen 1999; Begenisich 2004). The properties of maxi-K channels are also characteristically unique: (i) strong time dependence of the currents, (ii) strong outward rectification regulated by intracellular Ca2+, (iii) inhibition by paxilline, and (iv) large single-channel conductance of 100C300 pS (Latorre 1989; Pallanck & Ganetzky, 1994; Nehrke 2003; Salkoff 2006) (150C200 pS in mouse parotid acinar cells; Nehrke 2003; Thompson & Begenisich, 2006). Another distinctive feature of the maxi-K channel is its inhibition by IK1 current activation, which was demonstrated in parotid acinar cells as well as in a heterologous expression system (Romanenko 2006; Thompson & Begenisich, 2006). The molecular identities of IK1 and maxi-K channels in parotid acinar cells were confirmed in mice in which the and genes, respectively, were disrupted (Begenisich 2004; Romanenko 2006). Surprisingly, loss of either IK1 or maxi-K channel expression did not impair parotid gland fluid secretion. However, disruptions of both the and genes produced a substantial (> 70%) reduction in secretion by mouse parotid glands (Romanenko.