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Giant cell myocarditis is a rare and highly lethal disease that is characterized by a rapidly progressive course of biventricular dysfunction. The authors present a case of giant cell myocarditis that presented with incessant ventricular tachycardia and cardiogenic shock in which clinical improvement was achieved with immunosuppressive therapy.

Background: The present experiments were designed to study the importance of the carboxy-terminus of HKalpha2, for both function and integrity of assembly with beta1-Na+,K+-ATPase., Methods: For this purpose, stop codons were created, by polymerase chain reaction (PCR), at different positions in the carboxy-terminus of HKalpha2. Subsequently, chimeras between HKalpha2 and the carboxy-terminus of alpha1-Na+,K+-ATPase or with the carboxy-terminus of the gastric H+,K+-ATPase were created. Human embryonic kidney HEK-293 cells were used as expression systems for functional studies using 86Rb+ uptake and alpha/beta assembly using specific antibodies., Results: The results demonstrate that the entire carboxy-terminus of HKalpha2 is required for optimal protection of the alpha/beta complex from degradation and for functionality as evidenced by 86Rb+ uptake. The results also demonstrate that there was flexibility in the sequence of the carboxy-terminus. The last two tyrosines (Y1035Y1036) of HKalpha2 could be mutated to alanines and the carboxy-terminus of HKalpha2 could be replaced by the carboxy-terminus of alpha1-Na+,K+-ATPase while preserving transport activity., Conclusion: The entire carboxy-terminus of HKalpha2 is required for stable assembly with beta1-Na+,K+-ATPase and functionality.

Background: Previous experiments from our laboratory have demonstrated that HKalpha(2) coimmunoprecipitated with beta(1)-Na(+),K(+)-ATPase. Although HKalpha(2) is expressed abundantly in the apical membrane of distal colon, the demonstration that beta(1) localizes to this same membrane in distal colon has not been demonstrated previously., Methods: Immunolocalization was performed in distal colon using a polyclonal antibody against HKalpha(2) and a monoclonal antibody against beta(1)., Results: The results demonstrate that HKalpha(2) localizes to the apical membrane. Two pools of beta(1)-Na(+),K(+)-ATPase were detected. The first localized to the apical membrane. The second pool was detected in the basolateral membrane when distal colon sections were deglycosylated with glycosidase F. Therefore, our results demonstrate that beta(1) localizes to the apical membrane with HKalpha(2), and supports the view that beta(1) is the physiologic beta-subunit for HKalpha(2). We tested, therefore, the efficiency of the two beta-subunits expressed in distal colon (beta(1) and beta(3)) to support the activity of HKalpha(2). Human embryonic kidney HEK-293 cells were transiently cotransfected with HKalpha(2) plus beta(1) or HKalpha(2) plus beta(3). Subsequently, (86)Rb(+)-uptake and plasma membrane localization were evaluated. The results demonstrate that both HKalpha(2)/beta(1) and HKalpha(2)/beta(3) support (86)Rb(+)-uptake. However, (86)Rb(+)-uptake measured in the cells cotransfected with HKalpha(2) plus beta(1) exceeded that measured in cells expressing HKalpha(2)/beta(3). Fluorescence microscopy using enhanced green fluorescent protein cloned at the amino-terminus of HKalpha(2) demonstrated protein migration to the plasma membrane in cells cotransfected with EGFP-HKalpha(2) plus beta(1). In contrast, in cells cotransfected with EGFP-HKalpha(2) plus beta(3), the vast majority of the protein remained confined to intracellular compartments. The significantly higher (86)Rb(+)-uptake corresponded to additional localization of HKalpha(2) to the plasma membrane when coexpressed with beta(1) compared to beta(3)., Conclusion: Taken together, these and previous results from our laboratory indicate that beta(1)-Na(+),K(+)-ATPase is likely to represent the most physiologic and efficient subunit for HKalpha(2) assembly in distal colon.

Background: The present experiments were designed to study the importance of the carboxy-terminus of colonic H(+), K(+)-ATPase alpha-subunit (HKalpha(2)), for both function as well as integrity of assembly with beta1-Na(+), K(+)-ATPase., Methods: For this purpose, a mutation of 84 amino acids in the carboxy-terminus was created (DeltaHKalpha(2)) and HEK-293 cells were used as expression systems for functional studies using (86)Rb(+)-uptake, coimmunoprecipitation using specific antibodies and fluorescence microscopy using green fluorescent protein., Results: The results demonstrate that comparable levels of expression of HKalpha(2) and DeltaHKalpha(2) mRNA were observed when cells were cotransfected with beta1 subunit. However, the abundance of expression of full length HKalpha(2) protein exceeded that of the truncated protein DeltaHKalpha(2). Ouabain-sensitive (86)Rb(+)-uptake was present only in cells cotransfected with HKalpha(2)/beta(1), indicating that the mutation was incapable of sustaining functionality. Coimmunoprecipitation experiments demonstrated that HKalpha(2) protein was immunoprecipitated more abundantly than DeltaHKalpha(2) when coexpressed with beta1. The use of sucrose gradients and green fluorescence protein immunofluorescence demonstrated that while the DeltaHKalpha(2)/beta(1) complex was confined to the endoplasmic reticulum, the HKalpha(2)/beta(1) complex translocated to the plasma membrane., Conclusion: Taken together, our results are consistent with the view that the carboxy-terminus of HKalpha(2) facilitates the proper folding of the HKalpha(2)/beta(1) complex allowing translocation of the heterodimer to the plasma membrane where potassium uptake occurs. Otherwise, the alpha/beta complex is destined for degradation.

Background: The ubiquitous Na+-pump (Na+,K+-ATPase) assembles as a heterodimer of composition alpha/beta in some nephron segments, while in other segments it may exist as a heterotrimer of composition alpha/beta/gamma. The gamma-subunit has been reported to increase the affinity of the Na+-pump for adenosine 5'-triphosphate (ATP), and decrease affinity for both Na+ and K+. The alpha-subunit of the colonic H+,K+-ATPase (cHK) shares 75% sequence similarity with alpha1-Na+,K+-ATPase (alpha1) and assembles with beta1-Na+,K+-ATPase (beta1) in distal colon and renal medulla. Differences in pharmacological properties have been ascribed to when heterologously expressed function has been compared to function in vitro. The purpose of this study was to determine if cHK might associate with the gamma-subunit of the Na+,K+-ATPase (gamma) as a possible explanation for these variations in function., Methods: An antibody specific for the gamma was used in coimmunoprecipitation experiments to determine if the gamma assembles stably in vitro with cHK and beta1 in rat renal medulla or distal colon., Results: Our results demonstrate that the gamma-subunit assembles specifically with the Na+-pump, but not with cHK. Furthermore, the gamma-subunit assembly was specific for rat kidney and was not observed in distal colon., Conclusion: Since the gamma-subunit did not assemble with the cHK/beta1 complex, gamma-subunit assembly cannot explain those variations in ex vivo and in vitro pharmacologic properties ascribed to cHK.

Unlabelled: Peptides derived from the human transferrin receptor stimulate endosomal acidification via a Gi-type protein., Background: Acidification of the endosomal compartment is a prerequisite for intracellular processing of endocytosed complexes. Endosomal acidification is accomplished by an H+-ATPase, in parallel with a Cl- conductance. Previous studies from our laboratory have demonstrated that endosomal acidification is modulated by a pertussis toxin-sensitive mechanism, suggesting that endosomal acidification could be regulated through a self-contained signal transduction pathway. This study was designed to test this hypothesis using the transferrin receptor as a model., Methods: Synthetic peptides corresponding to a region of the cytosolic domain of the transferrin receptor and containing a KPKR sequence were used to stimulate endosomal acidification in a G-protein-dependent manner., Results: Peptides activated the Gi, as evidenced by stimulation of the rate of GTPgammaS binding. A transferrin receptor peptide that lacked the KPKR sequence did not stimulate endosomal acidification and failed to promote GTPgammaS binding to Gi proteins., Conclusions: These results demonstrate that regulation of endosomal acidification can be achieved, in part, through a Gi-mediated signal transduction pathway. These findings suggest that regulation of endosomal acidification through such a pathway may facilitate intracellular processing of the transferrin receptor.

Adenosine is released from renal cells, and extracellular adenosine may influence the effects of ischemia on medullary tubule segments by altering ion transport or renal hemodynamics. While adenosine release and excretion are enhanced during renal ischemia, the specific sites of renal adenosine production have not been completely elucidated. In the present study, extracellular adenosine concentrations in suspensions of renal outer medulla and thick ascending limb segments were quantitated by reversed-phase high performance liquid chromatography. Media from other medullary (OM) suspensions incubated for 8 and 15 minutes at 0% oxygen contained significantly greater amounts of adenosine (1.404 +/- 0.21 and 2.034 +/- 0.27 ng/micrograms protein, respectively), when compared to values obtained from media of suspensions incubated for equivalent periods under non-hypoxic conditions (8, 20, and 95% oxygen), 0.78 +/- 0.05 (8 min) and 1.37 +/- 0.21 ng/micrograms protein (15 min). Similarly, adenosine release was greater in medullary thick ascending limb (mTAL) suspensions incubated for 8 minutes at 0% versus 8% oxygen (0.81 +/- 0.17 vs. 0.20 +/- 0.12 ng/micrograms protein, respectively). Moreover, the observed increase in adenosine release by thick ascending limbs at 0% oxygen could be inhibited completely by either furosemide or ouabain. These studies demonstrate that: 1) the renal medulla and medullary thick ascending limb are sites of adenosine release; 2) adenosine release by the mTAL is enhanced significantly during hypoxic conditions; and 3) the increased release of adenosine during hypoxia appears to be related to ion transport and oxidative metabolism, as the increased release was prevented by two disparate inhibitors of transport in this segment.

The effect of bilateral vasectomy on in situ pH in seminiferous tubules, initial segments, proximal caput, middle corpus and proximal cauda epididymides of the rat has been studied employing in vivo microelectrode techniques. After bilateral vasectomy of four weeks duration, a significant increase in acidity of luminal fluid in the initial segments of the caput epididymides was observed. By eight weeks post vasectomy, luminal pH in the initial segments was significantly more acid as compared to sham-operated control animals or four weeks vasectomy while, in contrast, luminal pH in the proximal cauda epididymides was significantly more alkaline. The alteration of luminal fluid pH in the initial segments and proximal cauda epididymides after bilateral vasectomy may be the result of impairment of acid-base, as well as water transport pathways.

Approximately 85% of the filtered bicarbonate load is reabsorbed in the proximal convoluted tubule. Transport in this segment displays saturation kinetics, and exhibits a higher capacity for reabsorption in the earliest portion. Reclamation of bicarbonate is highly regulated in the proximal tubule: an increase in luminal [HCO3-], flow rate and arterial PCO2 increase, while alkalinization of the peritubular surface inhibits bicarbonate absorption. Angiotensin II also appears to regulate bicarbonate transport, especially in the S1 segment. The majority of the filtered bicarbonate load which escapes reabsorption in the proximal tubule is reabsorbed in the thick ascending limb of Henle's loop. Bicarbonate reclamation in this segment is enhanced by luminal [HCO3-] and furosemide, and by chronic metabolic acidosis and increased dietary sodium intake. Amiloride, AVP and glucagon inhibit absorption in the thick ascending limb.

Increased renal nerve activity and sodium retention have been implicated in the development of hypertension in genetically transmitted forms of this disease. The present studies were designed to investigate the relationship between renal nerve integrity and renal proximal tubule (Na+, K+)-ATPase activity in spontaneously hypertensive rats (SHR). (Na+, K+)-ATPase activity of basolateral membranes (BLMs) enriched from proximal tubules of five-week-old SHR was greater, 328.6 +/- 18.9 nmol Pi/mg protein.min, than in age-matched genetic controls rats (Wistar-Kyoto, WKY, rats), 262.3 +/- 34.6 nmol Pi/mg protein.min (P less than 0.02). There was no detectable difference in (Na+, K+)-ATPase activity of 13-week-old SHR and WKY rats. Prior renal denervation was associated with a reduction in proximal tubule basolateral membrane (BLM) (Na+, K+)-ATPase activity, 316.8 +/- 23.8 to 223.1 +/- 23.9 nmol Pi/mg protein/min (P less than 0.02), in five-week SHR. However, denervation had no effect on renal (Na+, K+)-ATPase activity in either WKY rats, nor did sham-denervation in SHR. In addition, exogenous norepinephrine, 1 microM, produced a more pronounced stimulation of (Na+, K+)-ATPase activity in basolateral membranes from SHR as opposed to WKY controls (40.2% vs. 28.7%). Therefore, renal nerve integrity and exogenous catecholamines have a greater stimulatory influence on proximal tubule (Na+, K+)-ATPase activity in the early stages (prior to 5 weeks) of the development of hypertension in SHR than in age-matched WKY rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of the male contraceptive, alpha-chlorohydrin, on in situ pH in seminiferous tubules and epididymal duct of the rat has been studied employing in vivo microelectrode techniques. After eight days of low-dose alpha-chlorohydrin administration (15mg/kg/day), a significant increase in acidity of luminal fluid in seminiferous tubules, proximal caput, middle caput, and proximal cauda epididymidis was observed. Increased acidity in the testis and epididymis may play an important role in the antifertility effect of alpha-chlorohydrin.

A nitrogen-centered free radical was spin-trapped from superoxide-catalyzed oxidation of indolic compounds, using the spin-trap phenyl-t-butyl-nitrone. The hyperfine splitting constants observed were aN = 13.9 G, a beta N = 3.6 G and a beta H = 2.3 G. Incubation of various indolic compounds with goat lung microsomes showed that only 3-methylindole was able to generate a free radical in the NADPH-dependent microsomal system, as tested by spin-trapping. The splitting constants were the same as those seen with superoxide incubations with 3-methylindole. The study demonstrates the generality of formation of a nitrogen-centered free radical from various indolic compounds. Enzymatic radical formation from 3-methylindole suggests a microsomal-activated free radical mechanism for the specificity of 3-methylindole-induced pulmonary toxicity.

Previous micropuncture studies supporting hydrogen secretion as the mechanism of bicarbonate reabsorption have relied on the demonstration of an acid disequilibrium pH in the proximal tubule after systemic administration of a carbonic anhydrase inhibitor. Previous calculations of disequilibrium pH, however, have involved the necessary assumption that PCO2 in the proximal convoluted tubule was equal to arterial blood PCO2. This assumption can no longer be supported in view of the recent demonstration that the PCO2 in proximal and distal tubular fluid exceeded arterial blood by approximately 25 mm Hg. The purpose of the present study was to determine directly pH and PCO2 with microelectrodes in both the early and late segments of the accessible proximal tubule of nine Sprague-Dawley rats before and after administration of benzolamide (2.0 mg/kg/hr, i.v.). In the early proximal tubule, pH decreased significantly after benzolamide administration from 6.98 +/- 0.03 to 6.62 +/- 0.03 pH U (P less than 0.001), and PCO2 also decreased from 65.1 +/- 1.2 to 59.3 +/- 1.4 mm Hg (P less than 0.005). In the late proximal convoluted tubule, pH did not change after benzolamide (6.75 +/- 0.02 to 6.77 +/- 0.02), but PCO2 decreased significantly (64.3 +/- 1.5 to 57.7 +/- 1.6) (P less than 0.01). We conclude: (1) the fall in both pH and PCO2 in the early proximal tubule indicates that carbonic acid, not carbon dioxide accumulates after inhibition of luminal carbonic anhydrase; (2) although PCO2 also decreased in the late proximal tubule, unlike the early segment, pH was unchanged after benzolamide administration, perhaps as a result of increased bicarbonate delivery; and (3) PCO2 in vivo was significantly greater than was systemic arterial PCO2 before and after benzolamide administration in both the early and late proximal convoluted tubule. These findings lend support to the view that bicarbonate reabsorption in the proximal convoluted tubule occurs, in part, by hydrogen secretion.

Previous studies have suggested that acetate hemodialysis causes myocardial depression. This study examines the acute effects of hemodialysis using, alternately, bicarbonate and acetate in the dialysate, on cardiac function in ten patients. These patients were also studied during acetate dialysis using a large surface area (SA) dialyzer. Each patient was dialyzed for 4 hr with: (1) 1.0 m2 SA dialyzer and bicarbonate dialysate; (2) 1.0 m2 SA dialyzer and acetate bath, and; (3) 2.5 m2 SA dialyzer and acetate dialysate. All studies were performed during isovolemic dialysis to separate the effects of changes in cardiac filling volume with hemodialysis, from changes in myocardial contractility. Myocardial function, as assessed by pre- and postdialysis echocardiographically derived fractional shortening (Fs) and mean velocity of circumferential shortening (VCF), improved (P less than 0.05) to the same extent, after all three dialysis treatments. This occurred despite greater increases (P less than 0.002) in pH and bicarbonate after bicarbonate dialysis and decreases (P less than 0.05) in PO2, PCO2 and bicarbonate after acetate dialysis with 2.5 m2 SA dialyzer. These results indicate that diffusive dialysis with both acetate and bicarbonate dialysate improves myocardial function and do not support the view that acetate influx during dialysis can lead to myocardial depression.

Recent studies have supported the possibility that mechanisms other than alterations in transcapillary oncotic pressure may contribute to edema formation in nephrotic syndrome. In a patient with a discrete, partial obstruction to lymphatic flow in the left upper extremity, the authors determined the transcapillary oncotic pressure differential in the obstructed, more edematous extremity and the contralateral, unobstructed extremity. The results demonstrate a normal transcapillary oncotic pressure gradient in the unobstructed extremity, while in contrast, the gradient in the obstructed extremity was reduced. Clinically, the edema resolved completely in conjunction with resolution of the nephrotic syndrome. The authors conclude, therefore, that the obstructed extremity may be more susceptible to edema formation because of inability to increase lymphatic flow during periods of hypoalbuminemia to levels sufficient to reduce the interstitial oncotic pressure gradient. Furthermore, the normal gradient in the unobstructed extremity supports the view that other mechanisms, such as intrinsic alterations in renal sodium reabsorption, may be involved in edema formation because edema was present without demonstrable alterations in the transcapillary oncotic pressure differential and plasma volume was increased in this patient.