Separation of cardiac plasmalemma into cell surface and T-tubular components. Distribution of saxitoxin- and nitrendipine-binding sites

To compare surface sarcolemmal with T-tubular distributions of [3H]saxitoxin (STX)- and [3H]nitrendipine (NTD)-binding sites, we centrifuged membrane vesicles from sheep and bovine ventricles on a 10-40% linear sucrose gradient from which fractions were assayed for STX and NTD binding; for markers of surface sarcolemma (ouabain-sensitive Na,K-ATPase activity, [3H]quinuclidinyl benzilate binding); and for markers of junctional sarcoplasmic reticulum known to be preferentially associated with T-tubules (ryanodine-sensitive Ca2+ uptake, calsequestrin, an Mr 300,000 putative phosphorylatable "foot" protein, and electron microscopically visible junctional sarcoplasmic reticulum-plasmalemma complexes). We identified three distinct peaks in the sucrose gradient, each characterized by significant high and low affinity STX- and high affinity NTD-binding: Peak I (approximately 19% sucrose), highly enriched in surface sarcolemma; Peak III (approximately 36% sucrose), enriched in junctional sarcoplasmic reticulum markers and hence in junctional sarcoplasmic reticulum complexes with T-tubule; and Peak II (approximately 27% sucrose), showing greatest specific STX binding and only moderate NTD binding, enriched in T-tubular membrane, unassociated with junctional sarcoplasmic reticulum. For ventricular myocytes, the ratio NTD sites/STX sites was 2.5 for surface sarcolemma, but only approximately 1.0 for T-tubules. Unlike data published for mammalian skeletal muscle, sheep and beef cardiac NTD receptors were not significantly more concentrated in T-tubular than in surface plasmalemma.     

Nerve growth factor treatment or cAMP elevation reduces Ca2+/calmodulin-dependent protein kinase III activity in PC12 cells

Ca2+/calmodulin-dependent protein kinase III (Ca2+/CaM kinase III) phosphorylates a protein of Mr = 100,000 (the 100-kDa protein), a major substrate for Ca2+/CaM-dependent protein phosphorylation found in many mammalian tissues and cell lines (Nairn, A.C., Baghat, B., and Palfrey, H.C. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 7939-7943). Treatment of PC12 cells with nerve growth factor (NGF) or forskolin resulted in a decrease in the depolarization-dependent phosphorylation of the 100-kDa protein in intact cells and in a decrease in the Ca2+/CaM-dependent phosphorylation of the 100-kDa protein in cytosolic extracts. In experiments using cytosolic extracts, the initial effect of NGF on the phosphorylation of the 100-kDa protein was observed in less than 1 h, was maximal (70% decrease) after 12 h, and began to recover after 24 h. The effect of forskolin was more rapid and the maximal effect was greater (90-95% decrease). Decreased Ca2+/CaM kinase III activity was also found in PC12 cells treated with epidermal growth factor, 2-chloroadenosine plus isobutylmethylxanthine, or dibutyryl cAMP. The effect of forskolin did not reverse unless it was removed. Cycloheximide blocked the recovery of Ca2+/CaM kinase III activity observed following the removal of forskolin but did not affect the ability of forskolin to reduce kinase activity. Short-term treatment with phorbol ester had little effect on Ca2+/CaM kinase III activity; long-term treatment with phorbol ester, which results in the disappearance of enzymatically detectable protein kinase C, had no effect on the ability of NGF or 2-chloroadenosine to reduce Ca2+/CaM kinase III activity. The level of the 100-kDa protein as determined by immunological techniques was not changed by any treatment. These results suggested that the effect of treatment of PC12 cells with NGF or forskolin was to reduce the level of Ca2+/CaM kinase III per se.     

Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.