Phosphatidylinositol transfer proteins (PITPs) have been shown to play important roles in regulating a number of signal transduction pathways that couple to vesicle trafficking reactions, phosphoinositide-driven receptor-mediated signaling cascades, and development. While yeast and metazoan PITPs have been analyzed in some detail, plant PITPs remain entirely uncharacterized. We report the identification and characterization of two soybean proteins, Ssh1p and Ssh2p, whose structural genes were recovered on the basis of their abilities to rescue the viability of PITP-deficient Saccharomyces cerevisiae strains. We demonstrate that, while both Ssh1p and Ssh2p share approximately 25% primary sequence identity with yeast PITP, these proteins exhibit biochemical properties that diverge from those of the known PITPs. Ssh1p and Ssh2p represent high-affinity phosphoinositide binding proteins that are distinguished from each other both on the basis of their phospholipid binding specificities and by their substantially non-overlapping patterns of expression in the soybean plant. Finally, we show that Ssh1p is phosphorylated in response to various environmental stress conditions, including hyperosmotic stress. We suggest that Ssh1p may function as one component of a stress response pathway that serves to protect the adult plant from osmotic insult.

Chronic exposure in a low-PO(2) environment (i.e., chronic hypoxia, CH) elicits an elevated hypoxic ventilatory response and increased hypoxic chemosensitivity in arterial chemoreceptors in the carotid body. In the present study, we examine the hypothesis that changes in chemosensitivity are mediated by endothelin (ET), a 21-amino-acid peptide, and ET(A) receptors, both of which are normally expressed by O(2)-sensitive type I cells. Immunocytochemical staining showed incremental increases in ET and ET(A) expression in type I cells after 3, 7, and 14 days of CH (380 Torr). Peptide and receptor upregulation was confirmed in quantitative RT-PCR assays conducted after 14 days of CH. In vitro recordings of carotid sinus nerve activity after in vivo exposure to CH for 1-16 days demonstrated a time-dependent increase in chemoreceptor activity evoked by acute hypoxia. In normal carotid body, the specific ET(A) antagonist BQ-123 (5 microM) inhibited 11% of the nerve discharge elicited by hypoxia, and after 3 days of CH the drug diminished the hypoxia-evoked discharge by 20% (P < 0.01). This inhibitory effect progressed to 45% at day 9 of CH and to nearly 50% after 12, 14, and 16 days of CH. Furthermore, in the presence of BQ-123, the magnitude of the activity evoked by hypoxia did not differ in normal vs. CH preparations, indicating that the increased activity was the result of endogenous ET acting on an increasing number of ET(A). Collectively, our data suggest that ET and ET(A) autoreceptors on O(2)-sensitive type I cells play a critical role in CH-induced increased chemosensitivity in the rat carotid body.

Recent studies have demonstrated that oxygen-sensitive type I cells in the carotid body express the gap junction-forming protein connexin43 (Cx43). In the present study, we examined the hypothesis that chronic exposure to hypoxia increases Cx43 expression in type I cells as well as in chemoafferent neurons in the petrosal ganglion. Immunocytochemical studies in tissues from normal rats revealed diffuse and granular Cx43-like immunoreactivity in the cytoplasm of type I cells and dense punctate spots of immunoreactive product at the margins of type I cells and near the borders of chemosensory cell lobules. Cx43-like immunoreactivity was not detectable in petrosal ganglion neurons from normal animals. After a 2-wk exposure to hypobaric (380 Torr) hypoxia, Cx43 immunostaining was substantially enhanced in and around type I cells. Moreover, chronic hypoxia elicited the expression of Cx43-like immunoreactivity in the cytoplasm of afferent neurons throughout the petrosal ganglion. Quantitative RT-PCR studies indicate that chronic hypoxia evokes a substantial increase in Cx43 mRNA levels in the carotid body, along with a marked elevation of Cx43 expression in the petrosal ganglion. Increased Cx43 expression and gap junction formation in type I cells and sensory neurons may contribute to carotid body adaptation during sustained stimulation in extreme physiological conditions.

In the present study, we have investigated the possible role of adenosine in the hypoxia-mediated increase in adenosine 3',5'-cyclic monophosphate (cAMP) in the carotid body. cAMP levels in rabbit carotid bodies superfused in vitro for 10 min were increased in the presence of adenosine (100 microM and 1.0 mM; maximum increase = 127%, P < 0.01). These effects were reduced by the nonspecific adenosine-receptor antagonist 1,3-dipropyl-8[p-sulfophenyl]xanthine (DPSPX; 10 microM). The specific A2-receptor agonist 2-[4'(2-carboxymethyl)phenylethylamino]-5'-N-ethylcarboxamido adenosine (CGS-21680; 100 nM) also elevated carotid body cAMP levels, an effect that was blocked by the specific A2-antagonist 3,7-dimethyl-L-propargyl-xanthine (DMPX; 50 microM). Hypoxia-evoked elevations in cAMP were potentiated in the presence of the adenosine-uptake inhibitor dipyridamole (100 nM) and blocked by exposure to adenosine-receptor antagonists. Our data suggest that the rabbit carotid body contains specific adenosine receptors (A2 subtype) that are positively coupled to adenylate cyclase and that increases in cAMP associated with hypoxia are mediated by the release of endogenous adenosine.

The influence of varying concentrations of a transmembrane peptide, gramicidin A (gA), and cholesterol (Chol) on the passive permeation of p-methylhippuric acid (MHA) and alpha-carbamoyl-p-methylhippuric acid (CMHA) across egg-lecithin membranes (EPC) has been investigated in vesicle efflux experiments. Incorporation of 0.25 volume fraction of gA in its nonchannel conformation increased the permeability coefficient (Pm) for CMHA by a factor of 6.0 +/- 1.8 but did not alter Pm for MHA, a more lipophilic permeant. In contrast, incorporation of 0.26 volume fraction Chol with no added protein decreased the Pm values for both CMHA and MHA by similar factors of 4.2 +/- 1.1 and 3.5 +/- 1.2, respectively. A quantitative structure-transport model has been developed to account for the dependence of Pm on the membrane concentrations of gA and Chol in terms of induced changes in both membrane chain ordering and hydrophobicity. Chain ordering is assumed to affect Pm for both permeants similarly since they are comparable in molecular size, while changes in Pm ratios in the presence of gA or Chol are attributed to alterations in membrane hydrophobicity. Changes in lipid chain ordering were detected by monitoring membrane fluidity using fluorescence anisotropy of 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene incorporated into the membranes. The influence of additives on membrane hydrophobicity, which governs Pm ratios through effects on solute partitioning into the barrier domain, were rationalized within the framework of regular solution theory using solubility parameters as a measure of membrane hydrophobicity. Fits of the Pm ratios using the theoretical model yielded solubility parameters for gA and Chol in EPC membranes of 13.2 and 7.7 (cal/ml)(1/2), respectively, suggesting that gA decreases the barrier domain hydrophobicity while Chol has a minimal effect on barrier hydrophobicity. After correcting for barrier domain hydrophobicity, permeability decrements due to membrane ordering induced by gA or Chol were found to exhibit a strong correlation with membrane order as predicted by free-surface-area theory, regardless of whether gA or Chol is used as the ordering agent.