Jugular venous `a'' wave in pulmonary hypertension: new insights from a Doppler echocardiographic study

Objective—To study the mechanisms underlying the dominant `a' wave seen in patients with primary pulmonary hypertension.

Design—Retrospective and prospective examination of the jugular venous pulse recording, flow in the superior vena cava, and Doppler echocardiographic studies.

Setting—A tertiary referral centre for both cardiac and pulmonary disease, with facilities for invasive and noninvasive investigation, and assessment for heart and heart-lung transplantation.

Patients—12 patients with primary pulmonary hypertension, most being considered for heart-lung transplantation.

Results—Two distinct patterns of venous pulse and superior vena caval flow were identified: a dominant `a' wave with no `v' wave, an absent or poorly developed `y' descent, and exclusively systolic downward flow in the superior vena cava (group 1, n = 8), and a dominant `v' wave, deep `y' descent and exclusively diastolic downward flow in the superior vena cava (group 2, n = 4). A comparison between the two groups showed age (mean (SD)) 42 (18) ν 36 (7) years, RR interval 700 (65) ν 740 (240) ms, left ventricular end diastolic dimension 3·6 (0·8) ν 3·2 (1·0) cm and end systolic dimension 2·1 (0·5) ν 2·3 (0·3) cm, right ventricular end diastolic dimension 2·6 (0·5) ν 2·8 (0·6) cm, and pressure drop between right ventricle and right atrium 60 (8) ν 70 (34) mm Hg to be similar. Duration of tricuspid regurgitation 520 (30) ν 420 (130) ms and the time interval of pulmonary closure to the end of the tricuspid regurgitant signal 140 (30) ν 110 (40) ms were longer in group 1 compared with group 2, whereas right ventricular filling time was much shorter 180 (70) ν 350 (130) ms. In seven patients from group 1, a single peak of forward tricuspid flow was present, but this pattern was seen in only one patient from group 2.

Conclusions—In patients with primary pulmonary hypertension, the apparent `a' wave seen in the venous pulse is, in fact, a summation wave. It is probably the result of large pressure changes that must underlie rapid acceleration and deceleration of blood across the tricuspid valve when the right ventricular filling time is short.

     

Nuclear magnetic resonance spectroscopy of excised human hearts

Background—Phosphorus nuclear magnetic resonance spectroscopy has been proposed as a method of studying the metabolism of the myocardium in patients. Little is known about ³¹P nuclear magnetic resonance spectroscopy of diseased human hearts.

Methods—Two donor hearts meeting the requirements for heart transplantation and 11 diseased hearts were removed during a transplantation procedure and were studied in a horizontal 2·35 T superconducting magnet. Spectra were obtained at 0°C about 30 minutes after the excision. The areas of the inorganic phosphate peak (Pi) and of the phosphocreatine peak (PCr) were summed and expressed as a ratio with respect to the area of the β ATP peak.

Results—The ratio (Pi + Pcr)/β ATP was found to be significantly lower in five hearts with a myocardial infarct (0·77 (0·18)) than in hearts with dilated cardiomyopathy (1·25 (0·29)) and in normal hearts (1·69 (0·11)). The area of the phosphodiester peak was expressed as a ratio with respect to the area of the β ATP peak: no differences were found between the three groups.

Conclusions—These results suggest that the phosphocreatine concentration is lower in ischaemic heart disease than in dilated cardiomyopathy and that the phosphodiester peak is probably not useful in distinguishing between these two types of heart disease.

     

Value of 17β-oestradiol in prevention of contracture formation

Akeson, W. H., Woo, S. L-Y., Amiel, D., Doty, D. H., and Rutherford, L. (1976).Annals of the Rheumatic Diseases, 35, 429-436. Value of 17β-oestradiol in prevention of contracture formation. Male and female rabbits were treated with 17β-oestradiol during the 9-week development of a contracture in the left hind limb. The right hind limb served as the paired control. Untreated (absolute control) animals received saline injections. After 9 weeks biochemical analyses of water, hexosamine, soluble and total collagen, as well as biomechanical measurements of joint stiffness, were performed on the dissected knees. In all cases 17β-oestradiol reduced the measured stiffness in the contractures by approximately 50% as compared to the contractures of the untreated animals. 17β-oestradiol, in addition, partially prevented the loss of water and hexosamine which occurs in untreated contractures. 17β-oestradiol administration also decreased the content of soluble collagen fractions in the periarticular connective tissue of both the control and experimental knees. The relationship of these biochemical findings to their mechanical consequences is discussed, and the results are related to a working hypothesis of stress- and motion-dependent homoeostasis within periarticular connective tissue.     

Blockade of the integrin alphaLbeta2 but not of integrins alpha4 and/or beta7 significantly prolongs intestinal allograft survival in mice

BACKGROUND—Small bowel transplantation remains a difficult therapeutic option endangered by a high rate of rejection and infectious complications. To improve these clinical results, it is mandatory to set up animal models to test alternative immunosuppressive regimens which may lead to immunotolerance.
AIMS—To determine the value of blockade of αLβ2 (LFA-1) and α4 and β7 integrins (α4β1, α4β7, and αEβ7) in the prevention of rejection of fetal small bowel grafts in mice and the effect of the association of calcineurin dependent drugs in anti-LFA-1 treated mice.
METHODS—Adult recipient mice engrafted with allogeneic fetal small bowel received a short course of anti-α4 and/or anti-LFA-1 monoclonal antibodies (mAb) with or without FK506 or cyclosporin A. In addition, in a set of experiment, β7−/− mice were used as recipients. Graft biopsies were performed and processed for standard histology.
RESULTS—Blockade of the pathways of the integrins α4 and β7 had a modest or no effect on intestinal graft survival. In contrast, transitory, short administration of anti-LFA-1 monoclonal antibody alone, when started before engraftment (day −1), allowed long term survival of intestinal grafts, even when associated with calcineurin dependent drugs. However, early withdrawal of FK506 reversed the immunosuppressive effect of anti-LFA-1 treatment.
CONCLUSION—These results suggest that firstly, anti-LFA-1, but not anti-α4 mAb treatment, may be useful in improving the results of intestinal transplantation, and secondly, that this treatment is not incompatible with long term administration of tacrolimus currently used in the prevention of small bowel graft rejection in humans.


Keywords: small bowel transplantation; integrins; calcineurin; tolerance; mouse     

Vasodilator myocardial perfusion imaging: demonstration of local electrophysiological changes of ischaemia

Objective—To examine the incidence and severity of myocardial ischaemia provoked in the course of perfusion scintigraphy by coronary vasodilators using endocardial recordings of steady state monophasic action potentials as an independent marker of early localised myocardial ischaemia.

Patients—31 men undergoing routine cardiac catheterisation for investigation of chest pain were studied.

Setting—A tertiary cardiac referral centre.

Design—Single site monophasic action potentials were recorded from the left or right ventricle or both (50 recording sites) during intravenous infusion of dipyridamole (0·015 mg/kg/min) for four minutes. Heart rate was held constant with atrial pacing at 20% above the patient's resting rate. Technetium-99m hexakis-2-methoxy-2-methylpropyl-isonitrile (MIBI) was administered four minutes after dipyridamole, and single photon emission tomographic imaging was performed an hour later. Rest images were obtained the next day (two day, two dose protocol). Recordings were divided into three groups based on the scintigraphic perfusion characteristics and coronary anatomical data for the action potential recording site: group 1—recordings from areas with a normal perfusion pattern (n = 30), group 2—recordings from areas with a perfusion defect and subtended by significantly narrowed coronary arteries without obvious angiographic collateral supply (n = 10), and group 3—recordings from areas with a perfusion defect and subtended by occluded arteries with angiographically evident collaterals from adjacent vessels (n = 10).

Results—There were changes in the duration of the monophasic action potential indicative of ischaemia—that is, shortening of duration of steady state action potential—in 18 of the 20 recordings from areas of abnormal perfusion. Peak changes were apparent eight minutes from the start of the dipyridamole infusion. Mean (SEM) values for duration of the action potential between control and peak effect at eight minutes were 276·5 (5·3) ms ν 276·6 (5·4) for group 1 (NS), 289·6 (4·7) ms ν 278·4 (4·9) ms for group 2 (p < 0·001), and 269·6 (5·7) ms ν 242·0 (4·4) for group 3 (p < 0·0001). These changes were significantly different between the three groups (p < 0·01). ST segment changes on the surface electrocardiogram were seen in only eight patients, all with areas of viable myocardium supplied by collateral vessels.

Conclusions—These data provide strong evidence for the presence of myocardial ischaemia in regions of reversible perfusion defects induced by dipyridamole. This study also shows that such ischaemia is more intense and more likely to be seen when myocardial viability is dependent on collateral circulation.

     

Polymorphisms in the tumour necrosis factor gene are not associated with severity of inflammatory polyarthritis

Background: Tumour necrosis factor alpha (TNFα) is a powerful inflammatory mediator in rheumatoid and other types of inflammatory arthritis. Polymorphisms within the TNFα gene have previously been investigated to determine their role in the aetiopathogenesis of rheumatoid arthritis (RA), but it is unclear whether reported associations are with susceptibility to, or severity of, disease.

Objective: To examine the association between both individual TNFα single nucleotide polymorphisms (SNPs) and haplotypes with the development and severity of erosions by 5 years in patients with inflammatory polyarthritis (IP).

Methods: 438 patients from the Norfolk Arthritis Register observational inception cohort of patients with IP were x rayed 5 years after disease onset. They were genotyped for nine SNPs mapping to the TNFα gene, using a SNaPshot primer extension assay. Haplotypes were constructed in patients with IP, who were compared for the presence and extent of erosions at 5 years.

Results: No association between individual TNFα SNPs or haplotypes in the patients who developed erosions at 5 years compared with those who remained non-erosive was found. Restricting analysis to patients who satisfied ACR criteria for RA by 5 years did not affect the conclusions.

Conclusion: The TNFα gene does not seem to be associated with severity as assessed by erosive outcome at 5 years in patients with IP.

     

Modification of the circadian rhythm of onset of acute myocardial infarction by long-term antianginal treatment

Objective—To elucidate the mechanism of the circadian pattern of onset of acute myocardial infarction by examining the effects of prior antianginal treatment upon it.

Design—Retrospective analysis of clock time of the onset of acute myocardial infarction by linear modelling to define the circadian distribution of hourly onset rates and to examine the deviation of treated groups of patients from this distribution.

Setting—Coronary care unit in a general hospital taking unselected acute admissions from a district of 0·9 million people.

Patients—A series of 2231 patients with confirmed acute myocardial infarction.

Results—A major 24 h cycle and smaller 12 h and 6 h cycles were present in patients not taking antianginal medication. Onset rates varied twofold over the day, with maxima around 10.00 am and 10.00 pm. This pattern was unchanged in patients on prior treatment with regular nitrates, but in those who had been taking a β blocker or a calcium antagonist the 24 h cycle was absent.

Conclusions—These results are best explained by the shared property of β blockers and calcium antagonists to reduce blood pressure and myocardial oxygen demand. The mid-morning peak of the onset of myocardial infarction is attributable to the physiological increase in sympathetic drive and cardiac work at that time. The data are not consistent with the triggering of the 24 h periodicity by fluctuations in coronary tone or haemostatic activity.

     

Long term follow up of long QT syndrome treated by overdrive pacing

Long term follow up of a patient with idiopathic long QT syndrome is described. A 5 year old girl was admitted with attacks of unconsciousness. Epilepsy was diagnosed and the patient was treated with anticonvulsants. During other episodes, ECG study showed torsades de pointes. The patient was treated with β blockers, stellectomy without success, and later with overdrive pacing. The young woman is now 43 years old and in good health. It is suggested that early overdrive pacing be implanted in young people with symptomatic long QT syndrome.


Keywords: long QT syndrome; β blockers; pacemaker; long term survival     

Left ventricular filling characteristics in pulmonary hypertension: a new mode of ventricular interaction

Objective—To examine the effects of pulmonary hypertension on left ventricular diastolic function and to relate the findings to possible mechanisms of interdependence between the right and left sides of the heart in ventricular disease.

Design—A retrospective and prospective analysis of echocardiographic and Doppler studies.

Setting—A tertiary referral centre for both cardiac and pulmonary disease.

Patients—29 patients with pulmonary hypertension (12 primary pulmonary hypertension, 10 pulmonary fibrosis, five atrial septal defect (ASD), and two scleroderma) were compared with a control group of 10 patients with an enlarged right ventricle but normal pulmonary artery pressure (six ASD, one after ASD closure, one ASD and pulmonary valvotomy, one tricuspid valve endocarditis and repair, and one pulmonary fibrosis). None had clinical or echocardiographic evidence of intrinsic left ventricular disease.

Main Outcome measures—M mode echocardiographic measurements were made of septal thickness, and left and right ventricular internal cavity dimensions. Doppler derived right ventricular to right atrial pressure drop, and time intervals were measured, as were isovolumic relaxation time, and Doppler left ventricular filling characteristics.

Results—The peak right ventricular to right atrial pressure gradient was (mean (SD)) 60 (16) mm Hg in pulmonary hypertensive patients, and 18 (5) mm Hg in controls. The time intervals P₂ to the end of the tricuspid regurgitation, and P₂ to the start of tricuspid flow were both prolonged in patients with pulmonary hypertension compared with controls (115 (60) and 120 (40) ν 40 (15) and 45 (10) ms, p values <0·001). Pulmonary hypertensive patients commonly had a dominant A wave on the transmitral Doppler (23/29); however, all the controls had a dominant E wave. Isovolumic relaxation time of the left ventricle was prolonged in pulmonary hypertensive patients compared with controls, measured as both A₂ to mitral valve opening (80 (25) ν 50 (15) ms) and as A₂ to the start of mitral flow (105 (30) ν 60 (15) ms, p values <0·001). The delay from mitral valve opening to the start of transmitral flow was longer in patients with pulmonary hypertension (30 (15) ms) compared with controls (10 (10) ms, p < 0·001). At the time of mitral opening there was a right ventricular to right atrial gradient of 12 (10) mm Hg in pulmonary hypertensive patients, but this was negligible in controls (0·4 (0·3) mm Hg, p < 0·001).

Conclusions—Prolonged decline of right ventricular tension, the direct result of severe pulmonary hypertension, may appear as prolonged tricuspid regurgitation. It persists until after mitral valve opening on the left side of the heart, where events during isovolumic relaxation are disorganised, and subsequent filling is impaired. These effects are likely to be mediated through the interventricular septum, and this right-left ventricular asynchrony may represent a hitherto unrecognised mode of ventricular interaction.

     

Early vasodilator treatment in myocardial infarction: appropriate for the majority or minority?

Objective—To assess the influence of vasodilator treatment started early after myocardial infarction on left ventricular size and function.

Setting—Coronary care unit, Royal Infirmary, Edinburgh.

Patients—105 patients with acute myocardial infarction (systolic blood pressure >90 mm Hg) were randomised within 24 hours of the start of pain. Unlike previous studies 88% of the patients received thrombolysis.

Methods—Double blind randomised placebo controlled study with either 12·5 mg of captopril three times daily or 20 mg of isosorbide mononitrate three times daily for 28 days.

Main outcome measures—Clinical outcome and left ventricular size and function assessed by echocardiography, radionuclide ventriculography, and magnetic resonance imaging.

Results—There was no difference in left ventricular size or function in either treatment group as measured one week after the end of the trial. Even the placebo group tended to decrease left ventricular diameter over the four week study period (one week: 5·0 (0·1) ν, five weeks: 4·8 (0·1) cm, NS). Four patients had an adverse clinical outcome in the placebo group whereas no adverse outcome was seen in the captopril group.

Conclusions—Vasodilator treatment may be of limited value or of no benefit for most infarct patients, particularly those treated with thrombolytic agents. Captopril, however, may benefit patients at high risk.

     

Plasma triglyceride and high density lipoprotein cholesterol as predictors of ischaemic heart disease in British men: The Caerphilly and Speedwell Collaborative Heart Disease Studies

Objective—To assess the roles of plasma triglyceride and high density lipoprotein (HDL) cholesterol concentrations in predicting ischaemic heart disease.

Design—Two prospective cohort studies with common core protocols.

Setting and participants—Both cohorts are 100% samples of middle aged men. In Caerphilly the 2512 men were living within a defined area. In Speedwell the 2348 men were registered with local general practitioners.

Main outcome measures—Fasting blood samples were taken at initial examination and plasma lipid concentrations were measured. Major ischaemic heart disease events were assessed from hospital notes, death certificates, and electrocardiograms.

Results—At first follow up, after an average of 5·1 years in Caerphilly and 3·2 years in Speedwell, 251 major ischaemic heart disease events had occurred. Men with triglyceride concentrations in the top 20% of the distribution had a relative odds value for ischaemic heart disease of 2·3 (95% confidence interval (95% CI) 1·3 to 4·1) compared with men in the bottom 20%, after adjusting for both plasma total and HDL cholesterol, and non-lipid risk factors. Men in the lowest 20% of the distribution of HDL cholesterol concentration had a relative odds value of 1·7 (95% CI 1·0 to 2·8) compared with the top 20%, after adjustment was made for total cholesterol and triglyceride concentrations, and non-lipid risk factors. These relations were not caused by β blockers, which were being taken by 5% of the men.

Conclusions—Plasma triglyceride concentration predicts major ischaemic events after allowance is made for total and HDL cholesterol concentrations and other risk factors. In these populations, triglyceride is a more important predictor than total cholesterol concentration.

     

Stoichiometry and arrangement of heteromeric olfactory cyclic nucleotide-gated ion channels

Native cylic nucleotide-gated (CNG) channels are composed of α and β subunits. Olfactory CNG channels were expressed from rat cDNA clones in Xenopus oocytes and studied in inside-out patches. Using tandem dimers composed of linked subunits, we investigated the stoichiometry and arrangement of the α and β subunits. Dimers contained three subunit types: α_wt, β_wt, and α_m. The α_m subunit lacks an amino-terminal domain that greatly influences gating, decreasing the apparent affinity of the channel for ligand by 9-fold, making it a reporter for inclusion in the tetramer. Homomeric channels from injection of α_wtα_wt dimers and from α_wt monomers were indistinguishable. Channels from injection of α_wtα_m dimers had apparent affinities 3-fold lower than α_wt homomultimers, suggesting a channel with two α_wt and two α_m subunits. Channels from coinjection of α_wtα_wt and ββ dimers were indistinguishable from those composed of α and β monomers and shared all of the characteristics of the α+β phenotype of heteromeric channels. Coinjection of α_wtα_m and ββ dimers yielded channels also of the α+β phenotype but with an apparent affinity 3-fold lower, indicating the presence of α_m in the tetramer and that α+β channels have adjacent α-subunits. To distinguish between an α-α-α-β and an α-α-β-β arrangement, we compared apparent affinities for channels from coinjection of α_wtα_wt and βα_wt or α_wtα_wt and βα_m dimers. These channels were indistinguishable. To further argue against an α-α-α-β arrangement, we quantitatively compared dose–response data for channels from coinjection of α_wtα_m and ββ dimers to those from α and β monomers. Taken together, our results are most consistent with an α-α-β-β arrangement for the heteromeric olfactory CNG channel.     

Expression of α and β subunits of the integrin superfamily in articular cartilage from macroscopically normal and osteoarthritic human femoral heads

OBJECTIVE—The objective of this study was to detail the topographical and zonal distribution of α and β subunits of the integrin superfamily in normal and osteoarthritic cartilage.
METHODS—Immunohistochemistry utilising antibodies towards α and β subunits was performed on cryostat sections of human articular cartilage from macroscopically normal (n = 6) and osteoarthritic (n = 6) femoral heads. Samples of articular cartilage were obtained from 12 topographically distinct sites from each femoral head. Each section was divided into zones (superficial, middle, deep) and staining scores were recorded.
RESULTS—Normal cartilage stained for integrin subunits α1, α5, αV, β1, β4, and β5, but not for α2, α3, α4, α6, β2, β3, and β6. Intact and non-intact residual cartilage from osteoarthritic femoral heads stained for α1, α2, α5, αV, β1, β4, and β5. Staining was occasionally seen for α4 and β2, but not for α3, α6, β3, and β6. There was no topographical variation in the staining for any of the subunits in either normal or osteoarthritic cartilage. The only subunit that displayed a zonal variation was αV; staining for this subunit was most pronounced in the superficial zone compared with the middle and deep zones.
CONCLUSION—Chondrocytes in normal and osteoarthritic cartilage express the integrin subunits α1, α5, αV, β1, β4, and β5. Chondrocytes in osteoarthritic cartilage, in addition, express the α2, α4, and β2 subunits. The αv subunit is expressed by more chondrocytes in the superficial zone in comparison with cells in the deeper zones. None of the subunits display topographical variation in expression.

Keywords: cartilage; integrins; immunohistochemistry; osteoarthritis     

DNA sequence analysis reveals extensive homologies of regions preceding hsp70 and alphabeta heat shock genes in Drosophila melanogaster

Two kinds of RNA are synthesized at the 87C1 chromosomal locus of Drosophila melanogaster in response to heat shock. One of these codes for the major heat shock protein, hsp70; the other, αβ RNA, derives from tandemly repeated αβ units consisting of adjacent α and β DNA elements and has no identified translation product. Another DNA element, γ, flanks the 5′ ends of some αβ units. Here we report the complete nucleotide sequence of the 617-base-pair α and the 733-base-pair γ element as well as a portion of the longer β element. Sequence comparisons between the γ element and the two hsp70 genes at 87C1 reveal that the 406 base pairs of γ immediately upstream from the 5′ end of the αβ unit exhibit 97.5% homology with the sequences at and upstream from the 5′ end of the hsp70 genes. A similar homology also exists between γ and an hsp70 gene present at another heat shock locus, 87A7, which contains no αβ units. These results, in conjunction with previous observations, strongly suggest that the coordinate induction by heat shock of the hsp70 and αβ genes is a consequence of their homologous 5′ flanking sequences. We propose that this extraordinary degree of sequence conservation stems from the recent transposition of αβ DNA to the 87C1 locus, an event that brought αβ sequences adjacent to, and under the regulation of, the hsp70 control element.     

Decay of prepulse facilitation of N type calcium channels during G protein inhibition is consistent with binding of a single Gβγ subunit

We have examined the modulation of cloned and stably expressed rat brain N type calcium channels (α_1B + β_1b + α₂δ subunits) by exogenously applied purified G protein βγ subunits. In the absence of G_βγ, barium currents through N type channels are unaffected by application of strong depolarizing prepulses. In contrast, inclusion of purified G_βγ in the patch pipette results in N type currents that initially facilitated upon application of positive prepulses followed by rapid reinhibition. Examination of the kinetics of G_βγ-dependent reinhibition showed that as the duration between the test pulse and the prepulse was increased, the degree of facilitation was attenuated in a monoexponential fashion. The time constant τ for the recovery from facilitation was sensitive to exogenous G_βγ, so that the inverse of τ linearly depended on the G_βγ concentration. Overall, the data are consistent with a model whereby a single G_βγ molecule dissociates from the channel during the prepulse, and that reassociation of G_βγ with the channel after the prepulse occurs as a bimolecular reaction.     

A Xenopus oocyte β subunit: Evidence for a role in the assembly/expression of voltage-gated calcium channels that is separate from its role as a regulatory subunit

Two closely related β subunit mRNAs (xo28 and xo32) were identified in Xenopus oocytes by molecular cloning. One or both appear to be expressed as active proteins, because: (i) injection of Xenopus β antisense oligonucleotides, but not of sense or unrelated oligonucleotides, significantly reduced endogenous oocyte voltage-gated Ca²⁺ channel (VGCC) currents and obliterated VGCC currents that arise after injection of mammalian α₁ cRNAs (α_1C and α_1E); (ii) coinjection of a Xenopus β antisense oligonucleotide and excess rat β cRNA rescued expression of α₁ Ca²⁺ channel currents; and (iii) coinjection of mammalian α₁ cRNA with cRNA encoding either of the two Xenopus β subunits facilitated both activation and inactivation of Ca²⁺ channel currents by voltage, as happens with most mammalian β subunits. The Xenopus β subunit cDNAs (β3xo cDNAs) predict proteins of 484 aa that differ in only 22 aa and resemble most closely the sequence of the mammalian type 3 β subunit. We propose that “α₁ alone” channels are in fact tightly associated α₁β3xo channels, and that effects of exogenous β subunits are due to formation of higher-order [α₁β]β_n complexes with an unknown contribution of β3xo. It is thus possible that functional mammalian VGCCs, rather than having subunit composition α₁β, are [α₁β]β_n complexes that associate with α₂δ and, as appropriate, other tissue-specific accessory proteins. In support of this hypothesis, we discovered that the last 277-aa of α_1E have a β subunit binding domain. This β binding domain is distinct from the previously known interaction domain located between repeats I and II of calcium channel α₁ subunits.     

The α2δ subunits of voltage-gated calcium channels form GPI-anchored proteins,a posttranslational modification essential for function

Voltage-gated calcium channels are thought to exist in the plasma membrane as heteromeric proteins, in which the α1 subunit is associated with two auxiliary subunits, the intracellular β subunit and the α₂δ subunit; both of these subunits influence the trafficking and properties of Ca_V1 and Ca_V2 channels. The α₂δ subunits have been described as type I transmembrane proteins, because they have an N-terminal signal peptide and a C-terminal hydrophobic and potentially transmembrane region. However, because they have very short C-terminal cytoplasmic domains, we hypothesized that the α₂δ proteins might be associated with the plasma membrane through a glycosylphosphatidylinositol (GPI) anchor attached to δ rather than a transmembrane domain. Here, we provide biochemical, immunocytochemical, and mutational evidence to show that all of the α₂δ subunits studied, α₂δ-1, α₂δ-2, and α₂δ-3, show all of the properties expected of GPI-anchored proteins, both when heterologously expressed and in native tissues. They are substrates for prokaryotic phosphatidylinositol-phospholipase C (PI-PLC) and trypanosomal GPI-PLC, which release the α₂δ proteins from membranes and intact cells and expose a cross-reacting determinant epitope. PI-PLC does not affect control transmembrane or membrane-associated proteins. Furthermore, mutation of the predicted GPI-anchor sites markedly reduced plasma membrane and detergent-resistant membrane localization of α₂δ subunits. We also show that GPI anchoring of α₂δ subunits is necessary for their function to enhance calcium currents, and PI-PLC treatment only reduces calcium current density when α₂δ subunits are coexpressed. In conclusion, this study redefines our understanding of α₂δ subunits, both in terms of their role in calcium-channel function and other roles in synaptogenesis.     

Angiogenesis promoted by vascular endothelial growth factor: Regulation through α1β1 and α2β1 integrins

Vascular endothelial growth factor (VEGF), also known as vascular permeability factor, is a cytokine of central importance for the angiogenesis associated with cancers and other pathologies. Because angiogenesis often involves endothelial cell (EC) migration and proliferation within a collagen-rich extracellular matrix, we investigated the possibility that VEGF promotes neovascularization through regulation of collagen receptor expression. VEGF induced a 5- to 7-fold increase in dermal microvascular EC surface protein expression of two collagen receptors—the α₁β₁ and α₂β₁ integrins—through induction of mRNAs encoding the α₁ and α₂ subunits. In contrast, VEGF did not induce increased expression of the α₃β₁ integrin, which also has been implicated in collagen binding. Integrin α₁-blocking and α₂-blocking antibodies (Ab) each partially inhibited attachment of microvascular EC to collagen I, and α₁-blocking Ab also inhibited attachment to collagen IV and laminin-1. Induction of α₁β₁ and α₂β₁ expression by VEGF promoted cell spreading on collagen I gels which was abolished by a combination of α₁-blocking and α₂-blocking Abs. In vivo, a combination of α₁-blocking and α₂-blocking Abs markedly inhibited VEGF-driven angiogenesis; average cross-sectional area of individual new blood vessels was reduced 90% and average total new vascular area was reduced 82% without detectable effects on the pre-existing vasculature. These data indicate that induction of α₁β₁ and α₂β₁ expression by EC is an important mechanism by which VEGF promotes angiogenesis and that α₁β₁ and α₂β₁ antagonists may prove effective in inhibiting VEGF-driven angiogenesis in cancers and other important pathologies.     

Association of calcium channel α1S and β1a subunits is required for the targeting of β1a but not of α1S into skeletal muscle triads

The skeletal muscle L-type Ca²⁺ channel is a complex of five subunits that is specifically localized in the triad. Its primary function is the rapid activation of Ca²⁺ release from cytoplasmic stores in a process called excitation-contraction coupling. To study the role of α_1S–β_1a interactions in the incorporation of the functional channel complex into the triad, α_1S and β_1a [or a β_1a-green fluorescent protein (GFP) fusion protein] were expressed alone and in combination in myotubes of the dysgenic cell line GLT. βGFP expressed in dysgenic myotubes that lack the skeletal muscle α_1S subunit was diffusely distributed in the cytoplasm. On coexpression with the α_1S subunit βGFP distribution became clustered and colocalized with α_1S immunofluorescence. Based on the colocalization of βGFP and α_1S with the ryanodine receptor the clusters were identified as T-tubule/sarcoplasmic reticulum junctions. Expression of α_1S with and without β_1a restored Ca²⁺ currents and depolarization-induced Ca²⁺ release. The translocation of βGFP from the cytoplasm into the junctions failed when βGFP was coexpressed with α_1S mutants in which the β interaction domain had been altered (α_1S-Y366S) or deleted (α_1S-Δ351–380). Although α_1S-Y366S did not associate with βGFP it was incorporated into the junctions, and it restored Ca²⁺ currents and depolarization-induced Ca²⁺ release. Thus, β_1a requires the association with the β interaction domain in the I–II cytoplasmic loop of α_1S for its own incorporation into triad junctions, but stable α_1S–β_1a association is not necessary for the targeting of α_1S into the triads or for its normal function in Ca²⁺ conductance and excitation-contraction coupling.