Clamping of Intracellular pH in Neurons from Neonatal Rat Brainstem during Hypercapnia

In this work, I have made attempts to clamp intracellular pH in the presence of hypercapnic acidosis (HA) in neurons from the locus coeruleus (LC) and nucleus of the solitary tract (NTS) in neonatal rat (ages P3 to P17) brainstem slices. Two approaches were used to minimize hypercapnia-induced ΔpHi: 1) an increase in intracellular buffering power with a high HEPES concentration using whole cell patching techniques in individual neurons, and 2) a weak acid diffusion technique that relies on an efflux of weak acid to counterbalance HA influx thereby clamping pHi in multiple neurons at once. pHi was measured using two pH-sensitive fluorescent dyes: membrane impermeable pyranine for the former approach, and the acetoxymethyl ester form of membrane permeable 2\u27,7\u27-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF) for the latter. Blunting with HEPES buffer was performed in the NTS only, with a calculated average percent blunting of hypercapnia-induced acidification of 73.4%. Experiments blunting via weak acid diffusion utilized an inverted microscope and two weak acids: acetic acid and caproic (hexanoic) acid. In NTS neurons (n=56), acetic acid blunted acidification by only 33.1% and in LC neurons by only 19.6% (n=52). Caproic acid blunted hypercapnia-induced acidification by 50.7% (n=58) and 45.8% (n=47) in NTS and LC neurons, respectively. Experiments were also repeated using an upright microscope. In these experiments, acidification was blunted by 45.8% (n=56) and 52.6% (n=52) in NTS and LC neurons, respectively. Concurrent influx of weak base, trimethyl amine, with HA was also used to blunt ΔpHi, but the results showed no blunting and, in fact, a greater acidification in response to HA: -18.1% in the NTS (n=40) and -27.8% in the LC (n=28). These techniques are clearly insufficient to accomplish a complete clamping of hypercapnia-induced changes in pHi. However, we have determined that the ability to clamp pHi is highly affected by diffusion of the weak acid both up to and into the cell. Blunting could be improved with better superfusion of slices and the use of more permeable weak acids. Overall, the ability to blunt ΔpHi, especially in numerous cells simultaneously, would be valuable in studying systems where pHi may play a role in cellular function, such as the involvement of pHi in chemosensitive signaling, bicarbonate reabsorption in the proximal tubule, free fatty acid diffusion in adipocytes, and acid-sensing taste receptors

Adolescent Male Human Papillomavirus Vaccination

Objective . To determine male vaccination rates with quadrivalent human papillomavirus vaccine (HPV4) before and after the October 2011 national recommendation to routinely immunize adolescent males. Methods . We reviewed HPV4 dose 1 (HPV4-1) uptake in 292 adolescent males in our urban clinic prior to national recommendations and followed-up for HPV4 series completion rates. After national recommendation, 248 urban clinic and 247 suburban clinic males were reviewed for HPV4-1 uptake. Factors associated with HPV4-1 refusal were determined with multiple logistic regression. Results . Of the initial 292 males, 78% received HPV4-1 and 38% received the 3-dose series. After recommendation, HPV4-1 uptake was 59% and 7% in urban and suburban clinics, respectively. Variables associated with HPV4-1 uptake/refusal included time period, race, type of insurance, and receipt of concurrent vaccines. Conclusions . HPV4-1 vaccination rates in our urban clinic were high before and after routine HPV vaccine recommendations for adolescent males. Our vaccination rates were much higher than in a suburban practice

pH Regulating Transporters in Neurons from Various Chemosensitive Brainstem Regions in Neonatal Rats

We studied the membrane transporters that mediate intracellular pH (pHi) recovery from acidification in brainstem neurons from chemosensitive regions of neonatal rats. Individual neurons within brainstem slices from the retrotrapezoid nucleus (RTN), the nucleus tractus solitarii (NTS), and the locus coeruleus (LC) were studied using a pH-sensitive fluorescent dye and fluorescence imaging microscopy. The rate of pHi recovery from an NH4Cl-induced acidification was measured, and the effects of inhibitors of various pH-regulating transporters determined. Hypercapnia (15% CO2) resulted in a maintained acidification in neurons from all three regions. Recovery in RTN neurons was nearly entirely eliminated by amiloride, an inhibitor of Na+/H+ exchange (NHE). Recovery in RTN neurons was blocked ∼50% by inhibitors of isoform 1 of NHE (NHE-1) but very little by an inhibitor of NHE-3 or by DIDS (an inhibitor of HCO3-dependent transport). In NTS neurons, amiloride blocked over 80% of the recovery, which was also blocked ∼65% by inhibitors of NHE-1 and 26% blocked by an inhibitor of NHE-3. Recovery in LC neurons, in contrast, was unaffected by amiloride or blockers of NHE isoforms but was dependent on Na+ and increased by external HCO3−. On the basis of these findings, pHi recovery from acidification appears to be largely mediated by NHE-1 in RTN neurons, by NHE-1 and NHE-3 in NTS neurons, and by a Na- and HCO3-dependent transporter in LC neurons. Thus, pHirecovery is mediated by different pH-regulating transporters in neurons from different chemosensitive regions, but recovery is suppressed by hypercapnia in all of the neurons

pH regulating transporters in neurons from various chemosensitive brainstem regions in neonatal rats

We studied the membrane transporters that mediate intracellular pH (pHi) recovery from acidification in brainstem neurons from chemosensitive regions of neonatal rats. Individual neurons within brainstem slices from the retrotrapezoid nucleus (RTN), the nucleus tractus solitarii (NTS), and the locus coeruleus (LC) were studied using a pH-sensitive fluorescent dye and fluorescence imaging microscopy. The rate of pHi recovery from an NH4Cl-induced acidification was measured, and the effects of inhibitors of various pH-regulating transporters determined. Hypercapnia (15% CO2) resulted in a maintained acidification in neurons from all three regions. Recovery in RTN neurons was nearly entirely eliminated by amiloride, an inhibitor of Na+/H+ exchange (NHE). Recovery in RTN neurons was blocked ∼50% by inhibitors of isoform 1 of NHE (NHE-1) but very little by an inhibitor of NHE-3 or by DIDS (an inhibitor of HCO3-dependent transport). In NTS neurons, amiloride blocked over 80% of the recovery, which was also blocked ∼65% by inhibitors of NHE-1 and 26% blocked by an inhibitor of NHE-3. Recovery in LC neurons, in contrast, was unaffected by amiloride or blockers of NHE isoforms but was dependent on Na+ and increased by external HCO3−. On the basis of these findings, pHi recovery from acidification appears to be largely mediated by NHE-1 in RTN neurons, by NHE-1 and NHE-3 in NTS neurons, and by a Na- and HCO3-dependent transporter in LC neurons. Thus, pHi recovery is mediated by different pH-regulating transporters in neurons from different chemosensitive regions, but recovery is suppressed by hypercapnia in all of the neurons

pH Regulating Transporters in Neurons from Various Chemosensitive Brainstem Regions in Neonatal Rats

We studied the membrane transporters that mediate intracellular pH (pHi) recovery from acidification in brainstem neurons from chemosensitive regions of neonatal rats. Individual neurons within brainstem slices from the retrotrapezoid nucleus (RTN), the nucleus tractus solitarii (NTS), and the locus coeruleus (LC) were studied using a pH-sensitive fluorescent dye and fluorescence imaging microscopy. The rate of pHi recovery from an NH4Cl-induced acidification was measured, and the effects of inhibitors of various pH-regulating transporters determined. Hypercapnia (15% CO2) resulted in a maintained acidification in neurons from all three regions. Recovery in RTN neurons was nearly entirely eliminated by amiloride, an inhibitor of Na+/H+ exchange (NHE). Recovery in RTN neurons was blocked ∼50% by inhibitors of isoform 1 of NHE (NHE-1) but very little by an inhibitor of NHE-3 or by DIDS (an inhibitor of HCO3-dependent transport). In NTS neurons, amiloride blocked over 80% of the recovery, which was also blocked ∼65% by inhibitors of NHE-1 and 26% blocked by an inhibitor of NHE-3. Recovery in LC neurons, in contrast, was unaffected by amiloride or blockers of NHE isoforms but was dependent on Na+ and increased by external HCO3−. On the basis of these findings, pHi recovery from acidification appears to be largely mediated by NHE-1 in RTN neurons, by NHE-1 and NHE-3 in NTS neurons, and by a Na- and HCO3-dependent transporter in LC neurons. Thus, pHirecovery is mediated by different pH-regulating transporters in neurons from different chemosensitive regions, but recovery is suppressed by hypercapnia in all of the neurons

pH Regulating Transporters in Neurons from Various Chemosensitive Brainstem Regions in Neonatal Rats

We studied the membrane transporters that mediate intracellular pH (pHi) recovery from acidification in brainstem neurons from chemosensitive regions of neonatal rats. Individual neurons within brainstem slices from the retrotrapezoid nucleus (RTN), the nucleus tractus solitarii (NTS), and the locus coeruleus (LC) were studied using a pH-sensitive fluorescent dye and fluorescence imaging microscopy. The rate of pHi recovery from an NH4Cl-induced acidification was measured, and the effects of inhibitors of various pH-regulating transporters determined. Hypercapnia (15% CO2) resulted in a maintained acidification in neurons from all three regions. Recovery in RTN neurons was nearly entirely eliminated by amiloride, an inhibitor of Na+/H+ exchange (NHE). Recovery in RTN neurons was blocked ∼50% by inhibitors of isoform 1 of NHE (NHE-1) but very little by an inhibitor of NHE-3 or by DIDS (an inhibitor of HCO3-dependent transport). In NTS neurons, amiloride blocked over 80% of the recovery, which was also blocked ∼65% by inhibitors of NHE-1 and 26% blocked by an inhibitor of NHE-3. Recovery in LC neurons, in contrast, was unaffected by amiloride or blockers of NHE isoforms but was dependent on Na+ and increased by external HCO3−. On the basis of these findings, pHi recovery from acidification appears to be largely mediated by NHE-1 in RTN neurons, by NHE-1 and NHE-3 in NTS neurons, and by a Na- and HCO3-dependent transporter in LC neurons. Thus, pHirecovery is mediated by different pH-regulating transporters in neurons from different chemosensitive regions, but recovery is suppressed by hypercapnia in all of the neurons