Ca2+ channel antagonist actions in bladder smooth muscle: comparative pharmacologic and [3H]nitrendipine binding studies

The actions of a series of 15 Ca2+ channel antagonists including D-600, nifedipine, and diltiazem were examined against K+ depolarization and muscarinic receptor induced responses in guinea pig bladder smooth muscle. Responses of bladder are very dependent upon extracellular Ca2+ and sensitive to the Ca2+ channel antagonists, the tonic component more than the phasic component of response. Regardless of stimulant, K+ or methylfurmethide (MF), or component of response, the same rank order of antagonist activities is expressed, suggestive of a single structure-activity relationship and the existence of a single category of binding site which may, however, exist in several affinity states. High affinity binding of [3H]nitrendipine (KD = 1.1 X 10(-10) M) occurs in bladder membranes, and similar high affinity binding was found in microsomal preparations from other smooth muscles including guinea pig and rat lung, rat vas deferens, uterus, and stomach. [3H]nitrendipine binding in the bladder was sensitive to displacement by other 1,4-dihydropyridines, paralleling their pharmacologic activities and showing excellent agreement with binding data previously obtained for guinea pig ileal smooth muscle. Comparison of pharmacologic data for inhibition of K+- and MF-induced responses by a common series of Ca2+ channel antagonists in bladder and ileum revealed excellent correlations. Neither pharmacologic nor binding studies suggest significant differences in Ca2+ channel antagonist properties in smooth muscle from bladder and intestine.     

Scalp reconstruction with a prefabricated abdominal flap carried by the radial artery.

Custom prefabrication of free flaps provides an unlimited variety of applications, since flaps can be created with expendable tissues and without restriction to naturally occurring vascular territories. These principles also can be used to customize flaps that could not be completed by conventional means. We report a case of scalp reconstruction using a random-pattern abdominal flap in which a radial artery fascial flap was induced to serve as the vascular carrier. In addition to providing durable scalp coverage, the prefabricated free flap enabled salvage of an abdominal flap that would otherwise have been aborted after intermediate transfer to the forearm.     

Characterization of (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum by laser-excited europium luminescence

The molecular environment of Ca2+ translocating sites of skeletal muscle sarcoplasmic reticulum (SR) (Ca2+ + Mg2+)-ATPase has been studied by pulsed-laser excited luminescence of Eu3+ used as a Ca2+ analogue. Interaction of Eu3+ with SR was characterized by investigating its effect on partial reactions of the Ca2+ transport cycle. In native SR vesicles, Eu3+ was found to inhibit Ca2+ binding, phosphoenzyme formation, ATP hydrolysis activity and Ca2+ uptake in parallel fashion. The non-specific binding of Eu3+ to acidic phospholipids associated with the enzyme was prevented by purifying (Ca2+ + Mg2+)-ATPase and exchanging the endogenous lipids with a neutral phospholipid, dioleoylglycerophosphocholine. The results demonstrate that the observed inhibition of Ca2+ transport by Eu3+ is due to its binding to Ca2+ translocating sites. The 7F0----5D0 transition of Eu3+ bound to these sites was monitored. The non-Lorentzian nature of the excitation profile and a double-exponential fluorescence decay revealed the heterogeneity of the two sites. Measurement of fluorescence decay rates in H2O/D2O mixture buffers further distinguished the sites. The number of water molecules in the first co-ordination sphere of Eu3+ bound at transport sites were found to be 4 and 1.5. Addition of ATP reduced these numbers to zero and 0.6. These data show that the calcium ions in translocating sites are well enclosed by protein ligands and are further occluded down to zero or one water molecule of solvation during the transport process.     

Regulation of cardiac sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase

Summary The two high affinity calcium binding sites of the cardiac (Ca²⁺ + Mg²⁺)-ATPase have been identified with the use of Eu³⁺. Eu³⁺ competes for the two high affinity calcium sites on the enzyme. With the use of laser-pulsed fluorescent spectroscopy, the environment of the two sites appear to be heterogeneous and contain different numbers of H₂O molecules coordinated to the ion. The ion appears to be occluded even further in the presence of ATP. Using non-radiative energy transfer studies, we were able to estimate the distance between the two Ca²⁺ sites to be between 9.4 to 10.2 A in the presence of ATP. Finally, from the assumption that the calcium site must contain four carboxylic side chains to provide the 6–8 ligands needed to coordinate calcium, and based on our recently published data, we predict the peptidic backbone of the two sites.     

Inhibitory actions of a series of Ca2+ channel antagonists against agonist and K+ depolarization induced responses in smooth muscle: an assessment of selectivity of action

The inhibitory effects of the Ca2+ channel antagonists D-600, diltiazem, nifedipine and seven 1,4-dihydropyridine analogs of nifedipine against 80 mM K+ depolarization induced responses in guinea pig trachea, parenchyma, and pulmonary artery and rat renal and mesenteric artery preparations were determined. Together with similar data previously obtained for guinea pig ileum and bladder, these data permitted an assessment of tissue selectivity of action in smooth muscles of a series of Ca2+ channel antagonists under constant conditions (saline composition) and an identical challenge (K+ depolarization). Very similar rank orders of activity were expressed in all tissues suggesting that the same basic structure-activity relationship operates. However, the series of antagonists were significantly less active in respiratory smooth muscle than in other visceral or vascular smooth muscles. pA2 values for a series of 1,4-dihydropyridine antagonists measured in guinea pig taenia coli against Ca2+-induced responses in K+-depolarizing media correlated with mean inhibitory concentration values against K+-induced responses, suggesting that the latter were an appropriate measure of antagonist potency. pA2 values measured for nifedipine, D-600, and diltiazem against Ca2+-induced responses in taenia coli in the presence of a depolarizing K+ saline, or methylfurmethide, histamine, or 5-hydroxytryptamine did not differ, suggesting that the same channels were activated regardless of stimulant.     

Estimation of inter-binding-site distances in sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase using Eu(III) luminescence energy transfer

We have used several trivalent lanthanides as probes for the high-affinity Ca(II)-binding site of the Ca(II) + Mg(II)-ATPase of skeletal muscle sarcoplasmic reticulum. The luminescent probes Eu(III) and Tb(III) were excited directly with pulsed laser light and the energy transfer efficiencies to several lanthanide acceptors were measured, under conditions in which most donor-acceptor pair occupied high-affinity Ca(II) sites. We obtain an inter-ionic site distance of about 0.8-0.9 nm. Energy transfer measurements were also done with Eu(III) in at least one Ca(II) site and bidentate Cr-ATP complex at the ATP hydrolytic site. Quenching of Eu(III) luminescence by Cr-ATP was total under these conditions. We calculate an upper limit of 1.0 nm for the distance from the Ca(II) site(s) to the complexed Cr(III) ion at the hydrolytic site.     

Characterization of calciphorin, the low molecular weight calcium ionophore, from rat liver mitochondria

Calciphorin, the putative mitochondrial calcium ionophore from rat liver mitochondria, exhibits the inherent properties of the mitochondrial calcium transport system and is similar to the calf heart preparation reported earlier. The protein has a strong selectivity for Ca2+, and has a Kd for Ca2+ of 56.5 +/- 6.6 microM and 13.9 +/- 2.1 microM in organic extraction and flow dialysis experiments, respectively. Reduction of the contaminating lipids from 23 +/- 6.5 to 1.73 +/- 0.4 moles per mole protein does not alter the affinities, Ca2+/protein stoichiometry or selectivity for Ca2+.     

Purification and characterization of the 45,000-Dalton fragment from tryptic digestion of (Ca2+ + Mg2+)-adenosine triphosphatase of sarcoplasmic reticulum.

Tryptic digestion of (Ca2+ + Mg2+)-ATPase from sarcoplasmic reticulum of rabbit skeletal muscle has previously been shown to cleave the enzyme initially into a 55,000-dalton fragment and a 45,000-dalton fragment. In the present study the two fragments are solubilized in sodium dodecyl sulfate (SDS) and separated by preparative polyacrylamide gel electrophoresis. The 45,000-dalton fragment is found to be a relatively nonselective, divalent cation-dependent ionophore when incorporated into an oxidized cholesterol membrane (BLM). Ionophoric activity of this fragment is inhibited by low concentrations of LaCl3, HgCl2, and various reducing agents. There appears to be one or two relatively inaccessible disulfide bonds in the 45,000-dalton fragment that are essential for transport. Addition of reducing agents inhibits the ionophoric activity of the succinylated undigested enzyme and the 45,000-dalton fragment, but has no effect on the 55,000-dalton fragment. These experiments imply that the 45,000-dalton fragment and the 55,000-dalton fragment are in a series arrangement in the membrane.     

A rapid method for purification of synthetic oligoribonucleotides.

A procedure for large-scale purification of synthetic oligoribonucleotides has been developed that has significant advantages over gel purification techniques currently in use. Synthesis was performed using commercially available 2'-O-silylated ribonucleoside 3'-O-phosphoramidites, and coupling efficiencies were consistently greater than 97% for oligoribonucleotides up to 31 residues in length. Using C4 reverse-phase chromatography to remove material not deprotected by treatment with tetrabutylammonium fluoride, we have eliminated reactants in which the 2'-O-silyl group is only partly removed, thus ensuring a homogeneous population of oligoribonucleotide.