Two functionally different Na/K pumps in cardiac ventricular myocytes

The whole-cell patch-clamp technique was used to voltage clamp acutely isolated myocytes at -60 mV and study effects of ionic environment on Na/K pump activity. In quiescent guinea pig myocytes, normal intracellular Na+ is approximately 6 mM, which gives a total pump current of 0.25 +/- 0.09 pA/pF, and an inward background sodium current of 0.75 +/- 0.26 pA/pF. The average capacitance of a cell is 189 +/- 61 pF. Our main conclusion is the total Na/K pump current comprises currents from two different types of pumps, whose functional responses to the extracellular environment are different. Pump current was reversibly blocked with two affinities by extracellular dihydro-ouabain (DHO). We determined dissociation constants of 72 microM for low affinity (type-1) pumps and 0.75 microM for high affinity (type-h) pumps. These dissociation constants did not detectably change with two intracellular Na+ concentrations, one saturating and one near half-saturating, and with two extracellular K+ concentrations of 4.6 and 1.0 mM. Ion effects on type-h pumps were therefore measured using 5 microM DHO and on total pump current using 1 mM DHO. Extracellular K+ half-maximally activated the type-h pumps at 0.4 mM and the type-1 at 3.7 mM. Extracellular H+ blocked the type-1 pumps with half-maximal blockade at a pH of 7.71 whereas the type-h pumps were insensitive to extracellular pH. Both types of pumps responded similarly to changes in intracellular-Na+, with 9.6 mM causing half-maximal activation. Neither changes in intracellular pH between 6.0 and 7.2, nor concentrations of intracellular K+ of 140 mM or below, had any effect on either type of pump. The lack of any effect of intracellular K+ suggests the dissociation constants are in the molar range so this step in the pump cycle is not rate limiting under normal physiological conditions. Changes in intracellular-Na+ did not affect the half-maximal activation by extracellular K+, and vice versa. We found DHO-blockade of Na/K pump current in canine ventricular myocytes also occurred with two affinities, which are very similar to those from guinea pig myocytes or rat ventricular myocytes. In contrast, isolated canine Purkinje myocytes have predominantly the type-h pumps, insofar as DHO-blockade and extracellular K+ activation are much closer to our type-h results than type-1. These observations suggest for mammalian ventricular myocytes: (a) the presence of two types of Na/K pumps may be a general property. (b) Normal physiological variations in extracellular pH and K+ are important determinants of Na/K pump current. (c) Normal physiological variations in the intracellular environment affect Na/K pump current primarily via the Na+ concentration. Lastly, Na/K pump current appears to be specifically tailored for a tissue by expression of a mix of functionally different types of pumps.     

Morphological and biochemical characterization and analysis of apoptosis

Apoptosis is a form of programmed cell death serving physiologic and homeostatic functions. However, recent evidence implicating apoptosis in the etiology and pathophysiology of known human diseases, such as heart diseases, cancer, AIDS, and neurodegenerative and autoimmune diseases are continually surfacing. This has spawned the need for identifying which methods are the most effective and well accepted to decipher its presence in a variety of research settings. We have therefore detailed the morphology and biochemical features of apoptotic cell death, with an emphasis on discriminating it from necrosis. In addition, we describe specific and selective techniques which are optimal to target hallmark apoptotic features, such as microscopy, Annexin V labeling, in situ nick-end labeling (TUNEL), and DNA fragmentation analysis by gel electrophoresis and ELISA for oligonucleosome-sized DNA. The advantages and disadvantages of each technique are discussed, as well as their experimental importance relative to one another. The methods have been described in a stepwise fashion, and can readily be applied in the majority of cell systems. Whether working on the tissue or single cell level, these methods are highly effective in qualifying and quantifying apoptosis. The application of these methods in conjunction with molecular techniques can further delineate the underlying mechanisms of apoptosis.     

Drug release from hydrophilic matrices. 1. New scaling laws for predicting polymer and drug release based on the polymer disentanglement concentration and the diffusion layer

Two scaling laws for predicting polymer and drug release profiles from hydrophilic matrices were developed. They were developed on the basis of the diffusion layer and the polymer disentanglement concentration, rho p,dis, the critical polymer concentration below which polymer chains detach off a gelled matrix that is undergoing simultaneous swelling and dissolution. The relation between rho p,dis and molecular weight, M1 for (hydroxypropyl)methylcellulose (HPMC) in water was established as rho p,dis (g/mL) varies M-0.8. This power-law relationship for rho p,dis, along with the diffusion layer adjacent to the gelled matrix, leads to the scaling law of mp(t)/mp(infinity) varies Meq-1.15, where mp(t)/mp(infinity) is the fractional HPMC release. The scaling law explains the observation that polymer and drug release rates decreased sharply with M at low M and approach limiting values at high M. Experimentally, mp(t)/mp(infinity) was found to scale with Meq as mp(t)/mp(infinity) varies Meq-0.93, where Meq is the equivalent matrix molecular weight. Moreover, fractional drug release, md(t)/md(infinity), followed Meq as md(t)/md(infinity) varies Meq-0.48. These two scaling laws imply that, if the release profiles are known for one composition, release profiles for other compositions can be predicted. The above two power laws lead to two master curves for mp(t)/mp(infinity) and md(t)/md(infinity), suggesting that the release mechanism for soluble drugs from HPMC matrices is independent of matrix compositions, presumably via a diffusion-controlled process. Limitations of the power laws are discussed.     

Pharmacology and intracellular signaling mechanisms of the native human orphan receptor BRS-3 in lung cancer cells

Neither the native ligand nor the cell biology of the bombesin (Bn)-related orphan receptor subtype 3 (BRS-3) is known. In this study, we used RT-PCR to identify two human lung cancer lines that contain sufficient numbers of native hBRS-3 to allow study: NCI-N417 and NCI-H720. In both cell lines, [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) stimulates [3H]inositol phosphate. In NCI-N417 cells, binding of 125I-[DTyr6,betaAla11,Phe13,Nle14]Bn(6-14) was saturable and high-affinity. [DPhe6,betaAla11,Phe13,Nle14]Bn(6-14) stimulated phospholipase D activity and a concentration-dependent release of [3H]inositol phosphate (EC50 = 25 nM) and intracellular calcium (EC50 = 14 nM); the increases in intracellular calcium were primarily from intracellular stores. hBRS-3 activation was not coupled to changes in adenylate cyclase activity, [3H]-thymidine incorporation or cell proliferation. No naturally occurring Bn-related peptides bound or activated the hBRS-3 with high affinity. Four different bombesin receptor antagonists inhibited increases in [3H]inositol phosphate. Using cytosensor microphysiometry, we found that [DPhe6,betaAla11,Phe13, Nle14]Bn(6-14) caused concentration-dependent acidification. The results show that native hBRS-3 receptors couple to phospholipases C and D but not to adenylate cyclase and that they stimulate mobilization of intracellular calcium and increase metabolism but not growth. The discovery of human cell lines with native, functional BRS-3 receptors, of new leads for a more hBRS-3-specific antagonist and of the validity of microphysiometry as an assay has yielded important tools that can be used for the identification of a native ligand for hBRS-3 and for the characterization of BRS-3-mediated biological responses.     

Impact of anti-vaccine movements on pertussis control: the untold story

To assess the impact of anti-vaccine movements that targeted pertussis whole-cell vaccines, we compared pertussis incidence in countries where high coverage with diphtheria-tetanus-pertussis vaccines (DTP) was maintained (Hungary, the former East Germany, Poland, and the USA) with countries where immunisation was disrupted by anti-vaccine movements (Sweden, Japan, UK, The Russian Federation, Ireland, Italy, the former West Germany, and Australia). Pertussis incidence was 10 to 100 times lower in countries where high vaccine coverage was maintained than in countries where immunisation programs were compromised by anti-vaccine movements. Comparisons of neighbouring countries with high and low vaccine coverage further underscore the efficacy of these vaccines. Given the safety and cost-effectiveness of whole-cell pertussis vaccines, our study shows that, far from being obsolete, these vaccines continue to have an important role in global immunisation.