Sexual dimorphism in rat left atrial function and response to adrenergic stimulation

A number of investigations in humans and animals suggest that there may be intrinsic sex-associated differences in cardiac function. Using left atrial preparations from male and female rat hearts, we examined differences in myocardial function and response to adrenergic agonists. Contractile parameters were measured in isolated atria by conventional isometric methods in the absence or presence of isoproterenol or phenylephrine. Responsiveness to Ca²⁺ was measured in detergent-skinned atrial fibers and actomyosin ATPase activity was measured in isolated myofibrils. Tetanic contractions were generated by treating the atrium with ryanodine followed by high frequency stimulation. Developed force was greater and maximal rates of contraction and relaxation were more rapid in the female atrium. The relationship between Ca²⁺ concentration and force in both intact atria and detergent-skinned atrial fibers in females fell to the left of that for males. At low Ca²⁺ concentrations, skinned fibers from female atria generated more force and myofibrils from female atria had higher myosin ATPase activity than males. Tetanic contraction in the presence of high extracellular Ca²⁺ was greater in female atria. Male atrium had larger inotropic responses to isoproterenol and to phenylephrine, but drug-elicited cAMP and inositol phosphate production did not differ between sexes. The results demonstrate sex-related differences in atrial function that can be partially explained by greater myofibrillar Ca²⁺-sensitivity in females. A potential contribution of sarcolemmal Ca2+ influx is suggested by greater tetanic contraction in ryanodine-treated female atrium. The larger response of males to adrenergic stimulation does not appear to be explained by higher production of relevant second messengers. Future studies will investigate the role of sex hormones in these sexually dimorphic responses and may indicate a need for gender-specific therapeutic interventions for myocardial dysfunction.     

Novel anti-bacterial activities of β-defensin 1 in human platelets: suppression of pathogen growth and signaling of neutrophil extracellular trap formation

Human β-defensins (hBD) are antimicrobial peptides that curb microbial activity. Although hBD's are primarily expressed by epithelial cells, we show that human platelets express hBD-1 that has both predicted and novel antibacterial activities. We observed that activated platelets surround Staphylococcus aureus (S. aureus), forcing the pathogens into clusters that have a reduced growth rate compared to S. aureus alone. Given the microbicidal activity of β-defensins, we determined whether hBD family members were present in platelets and found mRNA and protein for hBD-1. We also established that hBD-1 protein resided in extragranular cytoplasmic compartments of platelets. Consistent with this localization pattern, agonists that elicit granular secretion by platelets did not readily induce hBD-1 release. Nevertheless, platelets released hBD-1 when they were stimulated by α-toxin, a S. aureus product that permeabilizes target cells. Platelet-derived hBD-1 significantly impaired the growth of clinical strains of S. aureus. hBD-1 also induced robust neutrophil extracellular trap (NET) formation by target polymorphonuclear leukocytes (PMNs), which is a novel antimicrobial function of β-defensins that was not previously identified. Taken together, these data demonstrate that hBD-1 is a previously-unrecognized component of platelets that displays classic antimicrobial activity and, in addition, signals PMNs to extrude DNA lattices that capture and kill bacteria.     

Sex differences in expression of calcium-handling proteins and beta-adrenergic receptors in rat heart ventricle

Human studies reveal sex differences in myocardial function as well as in the incidence and manifestation of heart disease. Myocellular Ca(2+) cycling regulates normal contractile function; whereas cardiac dysfunction in heart failure has been associated with alterations in Ca(2+)-handling proteins. Beta-adrenergic receptor (beta-AR) signaling regulates activity of several Ca(2+)-handling proteins and alterations in beta-AR signaling are associated with heart disease. This study examines sex differences in expression of beta(1)-AR, beta(2)-AR, and Ca(2+)-handling proteins including: L-type calcium channel (Ca(v)1.2) , ryanodine calcium-release channels (RyR), sarcoplasmic reticular Ca(2+) ATPase (SERCA2), phospholamban (PLB) and Na(+)-Ca(2+) exchange protein (NCX) in healthy hearts from male and female Sprague-Dawley rats. Protein levels were examined using Western blot analysis. Abundance of mRNA was determined by real time RT-PCR normalized to abundance of GAPDH mRNA. Contraction parameters were measured in right ventricular papillary muscle in the presence and absence of isoproterenol. Results demonstrate that female ventricle has significantly higher levels of Ca(v)1.2, RyR, and NCX protein compared to males. Messenger RNA abundance for RyR, and NCX protein was significantly higher in females whereas Ca(v)1.2 mRNA was higher in males. No differences were detected in beta-ARs, SERCA2 or PLB. Female right papillary muscle had a faster maximal rate of force development and decline (+/- dF/dt). There were no sex differences in response to isoproterenol. Results show significant sex differences in expression of key ventricular Ca(2+)-handling proteins that are associated with small functional differences in +/- dF/dt. Further studies will determine whether differences in the abundance of these key proteins play a role in sex disparities in the incidence and manifestation of heart disease.     

Characterization of phospholipase C-mediated phosphatidylinositol degradation in rat heart ventricle

Phosphoinositide-specific phospholipase C (PI-PLC) activity was investigated in the rat heart ventricle. Incubation of ventricle homogenate or 100,000g supernatant fraction with [3H]myoinositol or [3H]arachidonate-labeled phosphatidylinositol in the presence of Ca2+ resulted in a decrease in phosphatidylinositol with a concomitant increase in water-soluble [3H]inositol phosphate or [3H]diglyceride, respectively. Total overt homogenate PI-PLC activity could be accounted for in the supernatant fraction. Neutral, zwitterionic, cationic, or anionic detergents did not unmask membrane-associated activity. While cytosolic phospholipase C was active against a pure phosphatidylinositol substrate in the presence of Ca2+, no hydrolytic activity was detected when phosphatidylinositol was presented as a component (4-5%) of a mixture of phospholipids. However, addition of deoxycholate to the incubation mixture (pH 6.5, Ca2+ 10(-3) M) containing mixed phospholipids resulted in the exclusive hydrolysis of inositol phospholipids. Ventricular supernatant phospholipase C-mediated phosphatidylinositol degradation has a sharp pH optimum at 5.5 and a specific requirement for Ca2+. Activity is maximal at 1 to 2 X 10(-3) M Ca2+, with inhibition occurring at higher levels. Under optimized conditions phosphatidylinositol is hydrolyzed at a rate of 20-25 nmol/min/mg protein. Multivalent cations inhibit Ca2+-dependent PI-PLC activity while monovalent cations and anions have no effect. There is no apparent selectivity for specific fatty acid moieties on phosphatidylinositol. Soluble PI-PLC is inhibited by sulfhydryl reagents, neomycin, mepacrine, trifluoperazine, and propranolol. Chlorpromazine, dibucaine, and tetracaine exert a biphasic influence, stimulating at lower and inhibiting at higher concentrations.     

Evaluation of protein <Emphasis Type="BoldItalic">in vitro</Emphasis> digestibility of <Emphasis Type="BoldItalic">Palmaria palmata</Emphasis> and <Emphasis Type="BoldItalic">Gracilaria verrucosa</Emphasis>

Palmaria palmata and Gracilaria verrucosa are edible red seaweeds and potential protein sources for human or animal nutrition, so studies were conducted on their in vitro protein digestibility. After 30 min predigestion by pepsin followed by 6 h digestion into a cell dialysis containing porcine pancreatin, the in vitro protein digestibility of P. palmata and G. verrucosa, expressed in regard to casein digestibility, was 4.9% and 42.1%, respectively. The level of protein digestibility seems to be related to the amount of soluble fibre, which was 45.3% and 30.5%, respectively.     

Characterization of phospholipase C-mediated polyphosphoinositide hydrolysis in rat heart ventricles

Phospholipase C (PLC)-mediated degradation of polyphosphoinositides (phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 4-phosphate (PIP] was found to be present in rat heart ventricular soluble and total membrane fractions (100,000g supernatant and pellet). Distribution of polyphosphoinositide-specific phospholipase C activity between the membrane and soluble fraction was approximately 63 and 33% of total activity, respectively, whereas, phosphatidylinositol (PI) degradation could be detected only in the soluble fraction. Optimal PIP2-PLC activity occurred at a pCa2+ of 4.5. A similar peak in PIP-PLC activity could be demonstrated in soluble and membrane preparations; however, the rate of PIP degradation in the soluble fraction continued to increase at the highest calcium level tested (pCa2+ 3). With the exception of Sr2+, other noncalcium polycations did not support homogenate PIP2-PLC activity. In the presence of Ca2+, addition of Mg2+, La3+, or Sr2+ (10(-3) M) inhibited PIP2-PLC while Mn2+ and Gd3+ stimulated activity. In both the total membrane and soluble fractions, maximal polyphosphoinositide degradation occurs at pH 5.5 and 6.8. The detergents deoxycholate, cholate, and saponin exert a biphasic effect on PIP2-PLC activity (stimulating at lower concentrations and inhibiting at higher concentrations). The deoxycholate effect is observed in both the cytosolic and membrane fractions. Neutral and cationic detergents inhibit PIP2-PLC activity in a concentration-dependent manner. Similar to cytosolic PI-PLC activity, PIP2-PLC appears to depend on intact sulfhydryl groups. In the presence of a mixture of all three inositol phospholipids or the three phosphoinositides plus noninositol phospholipids, polyphosphoinositides are preferentially degraded.     

Effect of estrogen on calcium-handling proteins, beta-adrenergic receptors, and function in rat heart

Regulation of cellular Ca(2+) cycling is central to myocardial contractile function. Loss of Ca(2+) regulation is associated with cardiac dysfunction and pathology. Estrogen has been shown to modify contractile function and to confer cardioprotection. Therefore, we investigated the effect of estrogen on expression of rat heart myocardial Ca(2+)-handling proteins and beta-adrenergic receptor (beta(1)-AR) and examined functional correlates. Female rats were sham-operated (SHAM) or ovariectomized. Two weeks after ovariectomy rats were injected (i.p.) daily with estradiol benozoate (OVX+EB) or sesame oil (OVX) for 2 weeks. Protein abundance was measured by immunoblotting and mRNA was quantified by real-time RT-PCR. OVX significantly decreased estrogen and progesterone levels and EB replacement returned both estrogen and progesterone to physiological levels. OVX induced a 75% reduction of uterine weight and a gain in body weight. Replacement restored weights to SHAM level. OVX increased and estrogen-replacement normalized abundance of beta(1)-AR and L-type Ca(2+) channel (Cav1.2) protein. OVX decreased sodium-Ca(2+) exchange protein (NCX) and estrogen restored protein abundance to SHAM levels. Sarcoplasmic reticular ATPase (SERCA), phospholamban (PLB), and ryanodine receptor (RyR) abundance was not altered by hormone status. Levels of mRNA encoding for beta(1)-AR, Cav1.2, and NCX were not influenced by OVX or estrogen replacement. OVX had no effect on SERCA and PLB mRNA level but estrogen replacement elicited a significant increase compared to OVX and SHAM. Estrogen-dependent changes in Ca(2+)-handling proteins and beta(1)-AR are theoretically consistent reduced myocellular Ca(2+) load. However, hormone-dependent alterations in protein were not associated with changes in contractile function.     

Platelet precursors display bipolar behavior

In this issue, Thon et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201006102) demonstrate that newly released platelets exhibit bipolar behavior, shifting back and forth between round cells and multibodied proplatelets (Thon et al., 2010). The authors define this intermediate as a preplatelet and, in doing so, shed new insight into the terminal steps of platelet maturation.     

Escaping the nuclear confines: signal-dependent pre-mRNA splicing in anucleate platelets

Platelets are specialized hemostatic cells that circulate in the blood as anucleate cytoplasts. We report that platelets unexpectedly possess a functional spliceosome, a complex that processes pre-mRNAs in the nuclei of other cell types. Spliceosome components are present in the cytoplasm of human megakaryocytes and in proplatelets that extend from megakaryocytes. Primary human platelets also contain essential spliceosome factors including small nuclear RNAs, splicing proteins, and endogenous pre-mRNAs. In response to integrin engagement and surface receptor activation, platelets precisely excise introns from interleukin-1beta pre-mRNA, yielding a mature message that is translated into protein. Signal-dependent splicing is a novel function of platelets that demonstrates remarkable specialization in the regulatory repertoire of this anucleate cell. While this mechanism may be unique to platelets, it also suggests previously unrecognized diversity regarding the functional roles of the spliceosome in eukaryotic cells.