Native smooth muscle L-type Ca v 1.2 calcium channels have been shown to support a fraction of Ca 2؉ currents with a window current that is close to resting potential. The smooth muscle L-type Ca 2؉ channels are also more susceptible to inhibition by dihydropyridines (DHPs) than the cardiac channels. It was hypothesized that smooth muscle Ca v 1.2 channels exhibiting hyperpolarized shift in steady-state inactivation would contribute to larger inhibition by DHP, in addition to structural differences of the channels generated by alternative splicing that modulate DHP sensitivities. In addition, it has also been shown that alternative splicing modulates DHP sensitivities by generating structural differences in the Ca v 1.2 channels. Here, we report a smooth muscle L-type Ca v 1.2 calcium channel splice variant, Ca v 1.2SM (1/8/9*/32/⌬33), that when expressed in HEK 293 cells display hyperpolarized shifts for steady-state inactivation and activation potentials when compared with the established Ca v 1.2b clone (1/8/9*/32/33). This variant activates from more negative potentials and generates a window current closer to resting membrane potential. We also identified the predominant cardiac isoform Ca v 1.2CM clone (1a/8a/⌬9*/32/33) that is different from the established Ca v 1.2a (1a/8a/⌬9*/31/33). Importantly, Ca v 1.2SM channels were shown to be more sensitive to nifedipine blockade than Ca v 1.2b and cardiac Ca v 1.2CM channels when currents were recorded in either 5 mM Ba 2؉ or 1.8 mM Ca 2؉ external solutions. This is the first time that a smooth muscle Ca v 1.2 splice variant has been identified functionally to possess biophysical property that can be linked to enhanced state-dependent block by DHP.

An estimate of up to 60% of genes are subjected to alternative splicing, and 15% of human genetic diseases are associated with mutation of the splice sites [Krawczak M, Reiss J, and Cooper DN. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum Genet 1992; 90: 41-54; Cooper TA, and Mattox W. The regulation of splice-site selection, and its role in human disease. Am J Hum Genet 1997; 61: 259-66; Modrek B and Lee CJ. Alternative splicing in the human, mouse and rat genomes is associated with an increased frequency of exon creation and/or loss. Nat Genet 2003; 34: 177-80; Modrek B, Resch A, Grasso C, and Lee C. Genome-wide detection of alternative splicing in expressed sequences of human genes. Nucleic Acids Res 2001; 29: 2850-9; Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, et al. Initial sequencing and analysis of the human genome. Nature 2001; 409: 860-921] . The molecular diversity of alternatively spliced transcripts provides templates for a myriad of protein structures that are potentially crucial to sustaining the complexity of human physiology. The extensive alternative splicing of the alpha(1)1.2-subunit of the L-type Ca(v)1.2 channel, producing splice variants with distinct electrophysiological and pharmacological properties, would impact directly on the function of the cardiovascular system. Cell-selective expression of Ca(v)1.2 channels containing a specific alternatively spliced exon increases the functional variations for specific cellular activities in response to changing physiological signals. However, the regulation or control of the alpha(1)1.2-subunit alternative splicing machinery is unknown, and the role of numerous splice variants expressed in a cell is a mystery. A systematic and concerted effort is required to determine all the possible combinations of alternatively spliced exons in alpha(1)1.2-subunits in smooth and cardiac muscles. This will provide useful information to monitor changes on the usage of the entire suite of alternatively spliced exons to help relate altered Ca(v)1.2 channel function to physiology and disease.

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The purpose of this study was to examine whether neutral endopeptidase and angiotensin I-converting enzyme, two membrane-bound metalloenzymes that are widely distributed in the microcirculation, play a role in bradykinin-induced increase in vascular permeability in the hamster cheek pouch. Changes in vascular permeability were quantified by counting the number of leaky sites and by calculating the clearance of fluorescein isothiocyanate (FITC)-dextran (molecular mass, 70,000 d) during suffusion of the cheek pouch with bradykinin. Bradykinin produced a concentration-and time-dependent increase in the number of leaky sites and clearance of FITC-dextran. The selective, active site-directed neutral endopeptidase inhibitors phosphoramidon (1.0 gM) and thiorphan (10.0 jiM) and the selective angiotensin I-converting enzyme inhibitor captopril (10.0 ,uM) each shifted the concentration-response curve to bradykinin significantly to the left. During suffusion with bradykinin (1.0 ,uM) and phosphoramidon, the number of leaky sites increased significantly from 17±2 to 27±4 sites per 0.11 cm2 (mean+SEM, p<0.05), and FITC-dextran clearance increased significantly from 1.0±0.2 to 2.1±0.3 ml/secxlO-6. During suffusion with bradykinin (1.0 gM) and captopril, the number of leaky sites increased significantly from 10±2 to 41±3 sites per 0.11 cm2, and FITC-dextran clearance increased significantly from 0.8±0.3 to 3.2±0.8 ml/secxlO-6. During suffusion with bradykinin (1.0 ,LM) and thiorphan, the number of leaky sites increased significantly from 15±3 to 47+7 sites per 0.11 cm2, and FITC-dextran clearance increased significantly from 0.8+0.2 to 4.7+0.6 ml/secxl106. Suffusion (NEP, EC 3.4.24.11) and angiotensin I-converting enzyme (ACE, EC 3.4.15.1), that hydrolyze bradykinin at the Pro7-Phe8 bond to inactive fragnents 1-7 and 8-9.2-4 The location of NEP and ACE in anatomic proximity to the receptors of bradykinin on postcapillary venular endothelial cells suggests that they may play an important role in modulating the edema-forming effects of the peptide in vivo. [2][3][4][5][6][7] We postulated that NEP and ACE each play an important role in modulating the edema-forming effects of bradykinin in vivo. We reasoned that if endogenous NEP and ACE degrade bradykinin to inactive fragments, then selective pharmacological inhibition of

The purpose of this study was to examine the effects of cigarette smoke extract on endothelium-dependent and endothelium-independent dilatation of arterioles in vivo. Using intravital microscopy, we measured diameter of arterioles contained within the microcirculation of the hamster cheek pouch during suffusion with acetylcholine and nitroglycerin, before and after treatment with cigarette smoke extract. Under control conditions, acetylcholine and nitroglycerin produced dose-related dilatation of cheek pouch arterioles. Superfusion of cigarette smoke extract (1.0%) did not alter baseline diameter of arterioles or vasodilatation in response to nitroglycerin but impaired dilatation of arterioles in response to acetylcholine. Next, we examined the possibility that impaired dilatation of cheek pouch arterioles in response to acetylcholine after exposure to cigarette smoke extract may be related to the release of substances produced via the cyclooxygenase pathway. In indomethacin-pretreated hamsters, acetylcholine produced similar vasodilatation before and after exposure to cigarette smoke extract. Thus these findings suggest that cigarette smoke extract impairs endothelium-dependent responses of cheek pouch arterioles. The mechanism of impaired responses of cheek pouch arterioles after exposure to cigarette smoke extract appears to be related to the release of substances produced via the cyclooxygenase pathway.