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R W Putnam

Publications and source records attributed to R W Putnam.

13 recordsLinked to original sources
PubMed1992

In vitro antimicrobial activity of amiloride analogs against Pseudomonas.

The effects of specific amiloride analogs on Na+ channel and Na+/H+ antiport function in eukaryotic cells have been well studied, but the effect of these agents on Pseudomonas is unknown. The antimicrobial activity of benzamil HCl, 5-(N-N-dimethyl)amiloride HCl (DMA), 5-(N,N-hexamethylene)amiloride HCl (HMA), and 5-(N-methyl-N-isobutyl)amiloride HCl (MIA) on 30 Pseudomonas strains (20 P. aeruginosa and 10 P. cepacia) were compared to amiloride HCl after a 24-hour incubation in Mueller-Hinton broth at 35 degrees C. At pH 7.3 the MIC range and MIC50 (in mg/l; MIC50 in parentheses) for amiloride HCl, benzamil HCl, DMA, HMA and MIA were 400 to > 800 (> 800), 200 to 800 (400), 200 to > 800 (400), 100 to 400 (200), and 100 to 400 (200), respectively, for P. aeruginosa and > 800 (> 800), 400 to > 800 (800), 400 to > 800 (800), 200 to 800 (200), and 200 to 800 (200), respectively, for P. cepacia. Alteration of pH from 5.5 to 8.5 had a slight effect on potency. We conclude that all the analogs studied were more potent antipseudomonal agents in vitro than amiloride, with the more lipophilic compounds HMA and MIA, having the most profound activity.

Amiloride↗
PubMed1991

Which value for the first dissociation constant of carbonic acid should be used in biological work?

The apparent first dissociation constant of carbonic acid has been defined in different ways in the literature. Harned and co-workers (8-10) have defined it in terms of molalities of the participating species, including H ions: Ks = mHmHCO3/mCO2. In contrast, Hastings and Sendroy have defined an apparent constant in which acidity is expressed as H ion activity: K'1 = aHmHCO3/mCO2. These constants differ by a factor gamma H, the activity coefficient of H ions at the prevailing ionic strength. Therefore, pK'1 is greater than pKs by an amount equal to -log gamma H, which, at mu = 0.16 M, is approximately 0.1. It is important that the correct value for the apparent dissociation constant or its logarithmic form be entered in the mass action expression or in the Henderson-Hasselbalch equation in order to prevent significant errors in the computation by means of these equations of quantities that cannot be directly measured. Specifically, for the derivation of bicarbonate concentration from PCO2 and pH (-log aH), pK'1 is to be used and not an uncorrected pKs.

Carbon Dioxide↗
PubMed1990

Effect of changes of pHi on intracellular calcium in a smooth muscle-like cell line.

The effect of changes of pHi on Cai were studied using fluorescent dyes in cells of the cultured smooth muscle-like line, BC3H-1. Resting Cai in these cells was 182 +/- 12 nM (n = 74) at pHo of 7.4. Upon exposure to NH4Cl, which rapidly alkalinized cells, a transient increase of Cai to 394 +/- 55 nM (n = 29) was observed. The peak of the transient occurred within 30 s of exposure to NH4Cl and returned to baseline within 1 minute. Two other procedures which resulted in rapid cellular alkalinization also caused a transient rise in Cai: exposure to and then removal of CO2 (Cai increased from 182 +/- 22 to 248 +/- 28 nM; n = 8); and exposure to and then removal of Na propionate (Cai increased from 242 +/- 32 to 456 +/- 71 nM; n = 9). The NH4Cl-induced Cai transient was eliminated by exposure to 0.2 mM TMB8 and to Ca-free solutions, but not by exposure to 0.5 mM LaCl3. Sustained changes of pHi can be induced by varying pHo. When pHo was lowered to 6.9, Cai fell by 49 +/- 11 nM but increased by 203 +/- 51 nM (n = 6) when pHo was raised to 7.9. These data indicate that rapid alkalinization of BC3H-1 cells results in a rapid transient rise of Cai. This transient is most likely due to the release of Ca from intracellular stores but may also involve an increase of Ca influx. Steady state values of Cai are positively correlated with steady state pHi. These data may have implications for the contractile state of smooth muscle during periods of acid/base disturbances and relate to the role of elevated pHi in cells from hypertensive animals.

Ammonium Chloride↗
PubMed1990

Steady-state pHi, buffering power, and effect of CO2 in a smooth muscle-like cell line.

Intracellular pH (pHi) was studied in the smooth muscle-like cell line, BC3H-1, using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). The initial pHi measured in 20 mM Na N-2-hydroxyethylpiperazine-N'-2 ethanesulfonic acid-buffered medium [NHB; external pH (pHo) 7.4, 37 degrees C] was 6.89 +/- 0.01 (n = 178). pHi was affected by changes in external pHo, pHi changing by approximately 70% of the change in pHo. The intrinsic buffering power (beta int) of these cells, measured either with NH4Cl or Na propionate pulses, is low for muscle cells, averaging approximately 10 mM/pH unit. Steady-state pHi of BC3H-1 cells in NHB acidified reversibly on exposure to 0.5 mM 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; 1.4 +/- 0.3 x 10(-4) pH/s), 1 mM amiloride (2.0 +/- 0.7 x 10(-4) pH/s), or Na-free solution (8.3 +/- 2.4 x 10(-4) pH/s) and alkalinized upon exposure to Cl-free solutions (9.7 +/- 2.2 x 10(-4) pH/s). Exposure of BC3H-1 cells to CO2-HCO3-buffered solutions resulted in a transient acidification followed by an alkalinization of 0.3-0.4 pH unit to a new steady-state pHi of 7.27 +/- 0.01 (n = 65). This new steady-state pHi acidified very slowly upon exposure to 1 mM amiloride (0.3 +/- 0.1 x 10(-4) pH/s), acidified more rapidly upon exposure to 0.5 mM DIDS (5.9 +/- 0.6 x 10(-4) pH/s) or Na-free solutions (9.8 +/- 1.0 x 10(-4) pH/s), and alkalinized on exposure to Cl-free solutions (24.5 +/- 1.3 x 10(-4) pH/s).(ABSTRACT TRUNCATED AT 250 WORDS)

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗
PubMed1990

pH regulatory transport systems in a smooth muscle-like cell line.

The membrane transport systems responsible for pH regulation in BC3H-1 cells were studied using the pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF). In nominally CO2-free Na N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer (NHB) recovery from acidification after an NH4Cl pulse was reversibly inhibited by 1 mM amiloride or by Na-free solutions. On exposure to 5% CO2-HCO3 (external pH constant at 7.4), BC3H-1 cells alkalinized by approximately 0.3-0.4 pH unit. This CO2-induced alkalinization was unaffected by 1 mM amiloride, markedly reduced by 0.5 mM 4-acetamido-4'-isothiocyanostilbene-2,2'-disulfonic acid (SITS), and inhibited by Na-free solutions. On readdition of Na, cells rapidly alkalinized, even in the presence of 1 mM amiloride. Exposure to Cl-free CO2-HCO3 solutions caused a rapid alkalinization of nearly 1 pH unit that was abolished by SITS, largely independent of Na, unaffected by amiloride, and unchanged by membrane depolarization in high external K solutions. CO2-induced alkalinization was slowed by approximately 75% after prolonged exposure of cells to Cl-free NHB, but a distinct recovery from acidification remained in these Cl-depleted cells. This recovery was Na-dependent, SITS-inhibitable, and unaffected by depolarization in high-K solutions. In the presence of CO2, the acidification seen in response to NH4Cl-induced alkalinization was reduced 50% by 0.5 mM SITS. These data suggest that the regulation of pH in BC3H-1 cells is mediated by at least three transport systems: 1) Na-H exchange; 2) Cl-HCO3 exchange; and 3) electroneutral (Na + HCO3)-Cl exchange.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗
PubMed1989

Thrombin induces a calcium transient that mediates an activation of the Na+/H+ exchanger in human fibroblasts.

The calcium dependence of growth factor-induced cytoplasmic alkalinization was determined in serum-deprived human fibroblasts (WS-1 cells). Intracellular pH (pHi) and intracellular calcium (Ca2+i) were measured using the fluorescent dyes 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein and fura2, respectively. Thrombin (10 nM) induced an alkalinization (0.18 +/- 0.01 pH units, n = 23) that was Na+-dependent and amiloride-sensitive, suggesting that the alkalinization was mediated by the Na+/H+ exchanger. Thrombin treatment caused a transient increase in Ca2+i (325 +/- 39 nM, n = 12) that preceded the observed increase in pHi. The increases in Ca2+i and pHi were dependent on the concentration of thrombin. The thrombin-induced increase in Ca2+i occurred in the absence of external calcium indicating that thrombin released calcium from internal stores. Inhibition of the thrombin-induced increase in Ca2+i with 8-diethylaminooctyl 3,4,5-trimethoxybenzoate hydrochloride or bis-(o-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid also inhibited the thrombin-stimulated increase in pHi. The calcium ionophore ionomycin was used to increase Ca2+i independent of growth factor stimulation. When Ca2+i was elevated with ionomycin, a concomitant increase in pHi was observed. The increase in pHi due to ionomycin was dependent on Na+ and sensitive to amiloride. The removal of external Ca2+i inhibited the ionomycin-induced elevation of both Ca2+i and pHi. The ionomycin-induced increases in Ca2+i and pHi were not inhibited by 8-diethylaminooctyl 3,4,5-trimethoxy-benzoate hydrochloride. The results suggest that thrombin treatment can activate the Na+/H+ exchanger, and this activation is mediated by an increase in Ca2+i.

Amiloride↗
PubMed1989

Physicians' perceptions of their role in cardiovascular risk reduction.

Fifty randomly selected family physicians were interviewed to evaluate the role of primary care physicians in the reduction and control of cardiovascular risk in their patients. The interview schedule, developed from the PRECEDE framework, incorporated three categories of factors modifying behavior: predisposing, reinforcing, and enabling. In relation to high blood pressure, elevated serum cholesterol, and smoking, physicians were questioned regarding (a) knowledge and beliefs of desirable practice, (b) perceptions of personal ability, (c) factors that affect their preventive performance, and (d) perceptions of their own role and those of other resources. Physicians believed all three risk factors to be modifiable, and that reduced cardiovascular risk could prolong life and improve quality of life. Participants perceived themselves most effective in reducing high blood pressure, followed by serum cholesterol reduction and smoking cessation. Relationships with patients, patient compliance, personal committment, and belief in the efficacy of risk reduction were most frequently perceived to contribute to effectiveness. Physicians perceived themselves least skilled in enhancing patient compliance and achieving behavior change. Most physicians were committed to cardiovascular disease prevention and saw their own role as central. Contributions of other resources were not well understood. The effect of these complex and interacting perceptions has implications for attempts to enhance physicians' preventive activities.

Cardiovascular Diseases↗
PubMed1985

Effect of insulin on intracellular pH in frog skeletal muscle fibers.

The effect of insulin on intracellular pH (pHi) and membrane potential was studied in frog semitendinosus muscle fibers, using recessed-tip pH-sensitive glass microelectrodes and conventional 3 M KCl-filled microelectrodes. After a lag period of approximately 20 min, insulin [1 mU/ml, 0.1% bovine serum albumin (BSA)] produced a slow hyperpolarization of 2-5 mV and an alkalinization of 0.05-0.10 pH unit, which were both completed within 1 h and were not reversed by washing in insulin-free solution for 1 h. The effect of insulin on the pHi recovery rate from CO2-induced acidification was examined at various membrane voltages. At normal membrane voltage, insulin (400 mU/ml, no BSA) slightly increased the slow pHi recovery (from 0.01 to 0.04 delta pH/h). In fibers depolarized in 15 mM K to about -50 mV, insulin nearly tripled the recovery rate (from 0.05 to 0.13 delta pH/h). This insulin-induced recovery was abolished by 1 mM amiloride, a Na-H exchange inhibitor. The increased pHi recovery in 15 mM K thus represents an increased Na-H exchange, which may be due to an interaction between insulin and either membrane depolarization, per se, or increased intracellular Ca. In fibers depolarized in 50 mM K to about -25 mV, insulin did not affect recovery (0.28 delta pH/h). This lack of insulin effect might be due to fiber swelling or to the difference in the time course of elevation of intracellular Ca at -25 and -50 mV. These results are consistent with an alkalinizing effect of insulin in frog muscle mediated by Na-H exchange.

Amiloride↗