Biomedical subjects

M Horiuchi

Publications and source records attributed to M Horiuchi.

At least 19 recordsLinked to original sources

PubMed2006

Relationship between oxygenation in inactive biceps brachii muscle and hyperventilation during leg cycling.

Inactive forearm muscle oxygenation has been reported to begin decreasing from the respiratory compensation point (RCP) during ramp leg cycling. From the RCP, hyperventilation occurs with a decrease in arterial CO2 pressure (PaCO2). The aim of this study was to determine which of these two factors, hyperventilation or decrease in PaCO2, is related to a decrease in inactive biceps brachii muscle oxygenation during leg cycling. Each subject (n = 7) performed a 6-min two-step leg cycling. The exercise intensity in the first step (3 min) was halfway between the ventilatory threshold and RCP (170+/-21 watts), while that in the second step (3 min) was halfway between the RCP and peak oxygen uptake (240+/-28 watts). The amount of hyperventilation and PaCO2 were calculated from gas parameters. The average cross correlation function in seven subjects between inactive muscle oxygenation and amount of hyperventilation showed a negative peak at the time shift of zero (r = -0.72, p<0.001), while that between inactive muscle oxygenation and calculated PaCO2 showed no peak near the time shift of zero. Thus, we concluded that decrease in oxygenation in inactive arm muscle is closely coupled with increase in the amount of hyperventilation.

Adult↗

PubMed2006

Interacting molecule of AT1 receptor, ATRAP, is colocalized with AT1 receptor in the mouse renal tubules.

The renin-angiotensin system in the kidney plays a critical role in the regulation of renal hemodynamics and sodium handling through the activation of vascular, glomerular and tubular angiotensin II type 1 (AT1) receptor-mediated signaling. We previously cloned a molecule that specifically bound to the AT1 receptor and modulated AT1 receptor signaling in vitro, which we named ATRAP (for AT1 receptor-associated protein). The purpose of this study is to analyze the renal distribution of ATRAP and to examine whether ATRAP is co-expressed with the AT1 receptor in the mouse kidney. We performed in situ hybridization, Western blot analysis, and immunohistochemistry to investigate the expression of ATRAP mRNA and protein in the mouse kidney. The results of Western blot analysis revealed the ATRAP protein to be abundantly expressed in the kidney. Employing in situ hybridization and immunohistochemistry, we found that both ATRAP mRNA and the protein were widely distributed along the renal tubules from Bowman's capsules to the inner medullary collecting ducts. ATRAP mRNA was also detected in the glomeruli, vasculature, and interstitial cells. In all tubular cells, the ATRAP protein colocalized with the AT1 receptor. Finally, we found that the dietary salt depletion significantly decreased the renal expression of ATRAP as well as AT1 receptor. These findings show ATRAP to be abundantly and broadly distributed in nephron segments where the AT1 receptor is expressed. Furthermore, this is the first report demonstrating a substantial colocalization of ATRAP and AT1 receptor in vivo.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Bovine posterior pituitary extract stimulates prolactin release from the anterior pituitary gland in vitro and in vivo in cattle.

It has been reported that the posterior pituitary (PP) gland contains a potent, unknown prolactin (PRL)-releasing factor (PRF) in rats. PRFs are assumed to be produced in neurones located within the hypothalamus, and to be peptidergic in nature. However, little is known about PRFs in domestic animals. To characterize the PRF in the PP of domestic animals, the present study examined the PRL-releasing activity of an acidic extract from bovine PP (bPP) in vitro and in vivo in cattle. First, the PRL-releasing effect of bPP extract was compared with that of PRL-releasing peptide (PrRP), and thyrotropin-releasing hormone (TRH) from cultured bovine anterior pituitary cells. The extract significantly increased PRL concentrations in the culture medium, at doses of 0.002 and 0.02 eq./ml (one eq. is the PP extract from one animal), compared with the control (p < 0.05). PrRP failed to stimulate the release of PRL. TRH significantly increased PRL concentrations in the culture medium, at doses from 10(-9) to 10(-7) M, compared with the control (p < 0.05). The rate of increase in the PRL concentration, by 0.02 eq./ml bPP extract, was significantly greater than that in TRH (p < 0.05). Secondly, plasma PRL responses to the intravenous (i.v.) injection of bPP extract (0.5 eq./head), PrRP [3.59 mug/kg body weight (BW)], TRH (1 mug/kg BW), and a dopamine receptor antagonist (sulpiride, 0.1 mg/kg BW), were examined in calves. PrRP failed to stimulate PRL release; however, plasma PRL increased immediately following the injection of bPP extract, TRH and sulpiride. The PRL-releasing effect of i.v. injections of TRH and sulpiride was more potent than that of bPP extract. Finally, plasma PRL responses to the intra-hypothalamic injection of bPP extract were examined in calves. The intra-hypothalamic infusion (arcuate nucleus) of 0.0625 eq./head of bPP extract strongly stimulated PRL release in calves (p < 0.05). The present results show that PP contains a physiologically potent PRF in cattle.

Animals↗

PubMed2005

Relationship between maximal oxygen uptake and oxygenation level in inactive muscle at exhaustion in incremental exercise in humans.

The aim of the present study was to determine whether the oxygenation level in an inactive muscle during an incremental exercise test, determined by near-infrared spectroscopy, influences the maximal oxygen uptake (Vo2max). The oxygenation level at the onset of incremental exercise was higher than that at rest and started to decrease at a high power output. A minimal level was observed at exhaustion during incremental exercise. Vo2 increased linearly after some delay, and the rate of increase in Vo2 was greater at a higher power output. Heart rate increased linearly after the time delay, and the rate of increase in heart rate did not change. There was a significant correlation between Vo2max and oxygenation level in inactive muscle at exhaustion (r=-0.89). We therefore concluded that the oxygenation level in inactive muscle at exhaustion during incremental exercise is associated with an individual difference in Vo2max.

Adult↗

PubMed2003

Effects of Ghrelin on growth hormone secretion from cultured adenohypophysial cells in pigs.

To clarify the direct effects of Ghrelin on growth hormone (GH) release from anterior pituitary (AP) cells in pigs, GH-releasing effects of human Ghrelin (hGhrelin) and rat Ghrelin (rGhrelin) on porcine AP cells were compared with GHRH in vitro. The AP cells were obtained from 6-month-old pigs and the cells (2 x 10(5) cells per well) were incubated for 2 h with the peptides after incubating in DMEM for 3 days. hGhrelin and rGhrelin significantly stimulated GH release from the cultured cells at doses of 10(-8) and 10(-7)M (P < 0.05). The rates of increase in GH at 10(-8) and 10(-7)M of hGhrelin were 82.7 and 131.9%, while those with rGhrelin were 43.9 and 79.5%, respectively. GHRH significantly stimulated GH release from the cells at a dose as low as 10(-11)M (P < 0.05), and the response to GHRH was greater than that induced by Ghrelins. In time-course experiments, GHRH continued to increase GH concentrations in media until 120 min after incubation; however, those in media treated with hGhrelin reached a plateau 60 min after incubation, and the maximal value was approximately one third that obtained with GHRH. When hGhrelin (10(-8)M) and GHRH (10(-8)M) were added together, additive effects of both peptides on the release of GH were observed (P < 0.05). Somatostatin (SS, 10(-7)M) significantly blunted GH release induced by hGhrelin (10(-8)M) and GHRH (10(-8)M) (P < 0.05). In the presence of SS, additive effects of hGhrelin and GHRH on the release of GH were observed (P < 0.05). These results show that Ghrelin directly stimulates GH release from anterior pituitary cells in pigs; however, the GH-releasing effect is weaker than that of GHRH in vitro. The present results also show that Ghrelin interacts with GHRH and SS to in the release of GH from porcine adenohypophysial cells.

Animals↗

PubMed2003

Experimental transmission of abnormal prion protein (PrPsc) in the small intestinal epithelial cells of neonatal mice.

Using an immunohistochemical method, we attempted to detect the transmission of abnormal prion protein (PrPsc) to the enterocytes of the small intestine of neonatal mice by oral exposure with sheep brain affected by scrapie. Five 1-day-old neonatal mice were exposed by oral inoculation to the homogenized brain of a scrapie-affected sheep. In the small intestine of all mice 1 hour after inoculation, immunoreactivity with antinormal prion protein (PrPc) antibody was seen in the cytoplasm of villus enterocytes. This finding suggests transmission of abnormal PrPsc into the cytoplasm of enterocytes. In control mice treated with normal sheep brain, no PrPc signal was seen in enterocytes of the small intestine. Immunopositivity for neurofilament protein and glial fibrillary acidic protein was seen in the cytoplasm of enterocytes of mice inoculated with scrapie and normal sheep brain. This suggests that the enterocytes of neonatal mice can absorb PrPsc and other macromolecular proteins of the sheep brain affected by scrapie and may be more important than previously thought as a pathway for PrPsc transmission in neonatal animals.

Animals↗

PubMed2002

Kinetics of CO(2) excessive expiration in constant-load exercise.

BACKGROUND: The purpose of this study was to examine the kinetics of excessively expired CO(2) (VCO(2) excess) in response to an increase in blood lactate (La) during constant-load exercise. METHODS: O(2) uptake (VO(2) ) and CO(2) output (VCO(2) ) were measured at exercise intensities of 55% and 70% of individual peak values of Vo2 obtained in incremental exercise. VCO(2) excess was obtained by multiplying the Vo2 obtained at 55% or 70% peakVoz by the difference between respiratory gas exchange ratios (VCO(2) /Vo(2) ) at 55% or 70% peak VO(2) and at 40% peakVO(2) . RESULTS: In both exercises of 55% and 70% peakVO(2) , VCO(2) excess remained constant for about the first minute, increased until about 2 min after the start of exercise, and then decreased. VCO(2) excess showed positive values even at 10 min after the start of exercise. The La level, on the other hand, increased during the first 5 min of exercise but did not show any significant change during the next 5 min. The values of VCO(2) excess were integrated from the start to the end of 10 min of exercise (CO(2) excess). CO(2) excess was found to be significantly related to the difference between La at 10 min after the start of exercise and at rest (DLa). End tidal CO(2) pressure (PETCO(2)) increased and then decreased in both exercises. The difference between PETCO(2) values at peak and at 10 min after the start of exercise (DPETCO(2) ) was significantly related to CO(2) excess. DPetco2 was also found to be significantly related to the increase in DLa. CONCLUSIONS: The kinetics of estimated VCO2excess is affected first by the increase in La and later by the decrease in PETCO(2) induced by the La increase.

Adult↗

PubMed2001

Roles of angiotensin II type 2 receptor stimulation associated with selective angiotensin II type 1 receptor blockade with valsartan in the improvement of inflammation-induced vascular injury.

BACKGROUND: To investigate the effect of angiotensin (Ang) II type 1 receptor (AT(1)) blocker on vascular remodeling and explore the possibility of the involvement of Ang II type 2 receptor (AT(2)) stimulation in this process, we examined the effects of the selective AT(1) blocker valsartan on the vascular injury in wild-type (Agtr2+) and AT(2)-null (Agtr2-) mice. METHODS AND RESULTS: Neointima formation and the proliferation of vascular smooth muscle cells (VSMCs) induced by cuff placement on the femoral artery were greater in Agtr2- mice than those in Agtr2+ mice. Treatment of mice with valsartan at a dose of 1 mg. kg(-1). d(-1), which did not influence systolic blood pressure, significantly decreased neointima formation and the proliferation of VSMCs, whereas the valsartan was less effective in Agtr2- mice. Moreover, cuff placement increased the expression of monocyte chemoattractant protein-1 (MCP-1); inflammatory cytokines such as tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-1beta; and infiltration of CD45-positive leukocytes and macrophages in the injured arteries and further enhanced them in Agtr2- mice, suggesting the antagonistic effects of AT(1) and AT(2) for vascular inflammation. Valsartan attenuated the expression of MCP-1, TNF-alpha, IL-6, IL-1beta, and infiltration of leukocytes and macrophages in the injured arteries; however, these effects of valsartan were less prominent in Agtr2- mice. CONCLUSIONS: These results suggest that the stimulation of the AT(2) receptor after AT(1) blockade is important in the improvement of the inflammatory vascular injury.

Angiotensin I↗

PubMed2001

Intracellular third loops in AT1 and AT2 receptors determine subtype specificity.

The recently cloned angiotensin II type 2 (AT2) receptor is a member of the seven transmembrane G-protein coupled receptor superfamily with a relatively low sequence homology with the angiotensin II type 1 (AT1) receptor subtype and counteracts the growth action of AT1 receptor. Intracellular third loops are known to be involved in interactions with various G proteins. We hypothesized that the intracellular third loop plays critical roles in determining the specificity of opposite functions of AT1 and AT2 receptor subtypes and examined this possibility using chimeric AT1 receptor, of which intracellular third loop is replaced with that of AT2 receptor. We transfected this chimeric receptor into PC 12 cells and observed that stimulation of this receptor inhibited extracellular signal-regulated kinase (ERK) activation and induces apoptosis, whereas the binding characteristics of this receptor remained those of ATI receptor. Taken together, these results support the notion that intracellular third loop is the critical determinant for mutually antagonistic AT1 and AT2 receptors' signaling pathways.

Animals↗

PubMed2001

Novel recognition mode between Vav and Grb2 SH3 domains.

Vav is a guanine nucleotide exchange factor for the Rho/Rac family that is expressed exclusively in hematopoietic cells. Growth factor receptor-bound protein 2 (Grb2) has been proposed to play important roles in the membrane localization and activation of Vav through dimerization of its C-terminal Src-homology 3 (SH3) domain (GrbS) and the N-terminal SH3 domain of Vav (VavS). The crystal structure of VavS complexed with GrbS has been solved. VavS is distinct from other SH3 domain proteins in that its binding site for proline-rich peptides is blocked by its own RT loop. One of the ends of the VavS beta-barrel forms a concave hydrophobic surface. The GrbS components make a contiguous complementary interface with the VavS surface. The binding site of GrbS for VavS partially overlaps with the canonical binding site for proline-rich peptides, but is definitely different. Mutations at the interface caused a decrease in the binding affinity of VavS for GrbS by 4- to 40-fold. The structure reveals how GrbS discriminates VavS specifically from other signaling molecules without binding to the proline-rich motif.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Effect of angiotensin II type 2 receptor on tyrosine kinase Pyk2 and c-Jun NH2-terminal kinase via SHP-1 tyrosine phosphatase activity: evidence from vascular-targeted transgenic mice of AT2 receptor.

Angiotensin II (Ang II) has two major receptor isoforms, AT1 and AT2. AT1 transphosphorylates Ca(2+)-sensitive tyrosine kinase Pyk2 to activate c-Jun NH2-terminal kinase (JNK). Although AT2 inactivates extracellular signal-regulated kinase (ERK) via tyrosine phosphatases (PTP), the action of AT2 on Pyk2 and JNK remains undefined. Using AT2-overexpressing vascular smooth muscle cells (AT2-VSMC) from AT2-transgenic mice, we studied these undefined actions of AT2. AT1-mediated JNK activity was increased 2.2-fold by AT2 inhibition, which was abolished by orthovanadate. AT2 did not affect AT1-mediated Pyk2 phosphorylation, but attenuated c-Jun mRNA accumulation by 32%. The activity of src-homology 2 domain-containing PTP (SHP-1) was significantly upregulated 1 min after AT2 stimulation. Stable overexpression of SHP-1 dominant negative mutant in AT2-VSMC completely abolished AT2-mediated inhibition of JNK activation and c-Jun expression. These findings suggest that AT2 inhibits JNK activity by affecting the downstream signal of Pyk2 in a SHP-1-dependent manner, leading to a decrease in c-Jun expression.

Angiotensin Receptor Antagonists↗

PubMed2001

Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes.

Na(+)/Cl(-)-dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [(35)S]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins. Thus, Na(+)/Cl(-)-dependent neurotransmitter transporters do not require oligomerization for substrate translocation.

Amino Acid Transport Systems, Neutral↗

PubMed2001

Inhibition of interactions and interconversions of prion protein isoforms by peptide fragments from the C-terminal folded domain.

The formation of protease-resistant prion protein (PrP-res or PrP(Sc)) involves selective interactions between PrP-res and its normal protease-sensitive counterpart, PrP-sen or PrP(C). Previous studies have shown that synthetic peptide fragments of the PrP sequence corresponding to residues 119-136 of hamster PrP (Ha119-136) can selectively block PrP-res formation in cell-free systems and scrapie-infected tissue culture cells. Here we show that two other peptides corresponding to residues 166-179 (Ha166-179) and 200-223 (Ha200-223) also potently inhibit the PrP-res induced cell-free conversion of PrP-sen to the protease-resistant state. In contrast, Ha121-141, Ha180-199, and Ha218-232 were much less effective as inhibitors. Mechanistic analyses indicated that Ha166-179, Ha200-223, and peptides containing residues 119-136 inhibit primarily by binding to PrP-sen and blocking its binding to PrP-res. Circular dichroism analyses indicated that Ha117-141 and Ha200-223, but not non-inhibitory peptides, readily formed high beta-sheet structures when placed under the conditions of the conversion reaction. We conclude that these inhibitory peptides may mimic contact surfaces between PrP-res and PrP-sen and thereby serve as models of potential therapeutic agents for transmissible spongiform encephalopathies.

Amino Acid Sequence↗

PubMed2001

Sulfated glycans and elevated temperature stimulate PrP(Sc)-dependent cell-free formation of protease-resistant prion protein.

A conformational conversion of the normal, protease- sensitive prion protein (PrP-sen or PrP(C)) to a protease-resistant form (PrP-res or PrP(Sc)) is commonly thought to be required in transmissible spongiform encephalopathies (TSEs). Endogenous sulfated glycosaminoglycans are associated with PrP-res deposits in vivo, suggesting that they may facilitate PrP-res formation. On the other hand, certain exogenous sulfated glycans can profoundly inhibit PrP-res accumulation and serve as prophylactic anti-TSE compounds in vivo. To investigate the seemingly paradoxical effects of sulfated glycans on PrP-res formation, we have assayed their direct effects on PrP conversion under physiologically compatible cell-free conditions. Heparan sulfate and pentosan polysulfate stimulated PrP-res formation. Conversion was stimulated further by increased temperature. Both elevated temperature and pentosan polysulfate promoted interspecies PrP conversion. Circular dichroism spectropolarimetry measurements showed that pentosan polysulfate induced a conformational change in PrP-sen that may potentiate its PrP-res-induced conversion. These results show that certain sulfated glycosaminoglycans can directly affect the PrP conversion reaction. Therefore, depending upon the circumstances, sulfated glycans may be either cofactors or inhibitors of this apparently pathogenic process.

Animals↗

PubMed2001

Polypeptide synthesis using an expressed peptide as a building block for condensation with a peptide thioester: application to the synthesis of phosphorylated p21Max protein(1-101).

An expressed peptide proved to be useful as a building block for the synthesis of a polypeptide via the thioester method. A partially protected peptide segment, for use as a C-terminal building block, could be prepared from a recombinant protein; its N-terminal amino acid residue was transaminated to an alpha-oxoacyl group, the side-chain amino groups were then protected with t-butoxycarbonyl (Boc) groups, and. finally, the alpha-oxoacyl group was removed. On the other hand, an O-phosphoserine-containing peptide thioester was synthesized via a solid-phase method using Boc chemistry. These building blocks were then condensed in the presence of silver ions and an active ester component. During the condensation, epimerization at the condensation site could be suppressed by the use of N,N-dimthylformamide (DMF) as a solvent. Using this strategy, a phosphorylated partial peptide of the p21Max protein, [Ser(PO3H2)2.11]-p21Max(1-101), was successfully synthesized.

Basic-Leucine Zipper Transcription Factors↗

PubMed2001

Simultaneous resection of pancreas and liver metastases from different metachronous primary cancers.

Resection of a pancreatic head tumor and partial resection of the liver for metastatic lesions were carried out simultaneously in a 72-year-old woman. The patient had a history of two previous operations, right nephrectomy for renal cell carcinoma (clear cell type), done 14 years previously, and an Autincloss procedure for cancer of the left breast (solid tubular carcinoma); (T1N0M0; stage I) done 7 years previously. At the current presentation, preoperative radiographic examination showed a hypervascular tumor in each of the pancreatic and hepatic lesions, but with different patterns. On the basis of histological findings in the two resected specimens, it was difficult to establish whether the hepatic tumor originated from the renal cell carcinoma or the breast cancer, but postoperative immunohistochemical studies for carcinoembryonic antigen (CEA), estrogen receptors, and gross cystic disease fluid protein (GCDFP)-15 showed that the pancreatic tumor had metastasized from the renal cell carcinoma, and the liver tumor from the breast cancer. The immunohistochemical investigation of different markers thus proved to be useful in making the final diagnosis of metastatic lesions from different and metachronous cancers.

Aged↗

PubMed2001

Pivotal role of tyrosine phosphatase SHP-1 in AT2 receptor-mediated apoptosis in rat fetal vascular smooth muscle cell.

OBJECTIVE: To examine the possible crosstalk and the roles of angiotensin (Ang) II type 1 (AT1) and type 2 (AT2) receptors in the control of apoptosis in fetal vascular smooth muscle cells (VSMCs). METHODS: Fetal VSMCs were prepared from rat fetal aorta at embryonic day 20. Expression of Ang II receptors was measured by a radioligand binding assay. Apoptotic changes were assessed by caspase 3 activity and chromatin dye staining. Regulation of extracellular signal-regulated kinase (ERK) activity via Ang II receptors was analysed by determining phosphorylated ERK with Western blot. Ang II receptor-mediated activation of tyrosine phosphatase SHP-1 was assessed by protein tyrosine phosphatase assay. RESULTS: The expression of AT1 and AT2 receptors was approximately 70%: 30% per cell. Serum depletion induced apoptosis in fetal VSMCs and selective AT1 receptor stimulation attenuated the apoptotic changes, whereas selective AT2 receptor activation enhanced apoptosis. Ang II increased ERK phosphorylation, which was inhibited by addition of the AT1 receptor-specific antagonist CV11974, but enhanced by addition of the AT2 receptor-specific antagonist PD123319, suggesting that activation of AT2 receptor attenuated the AT1 receptor-mediated ERK phosphorylation. Moreover, we demonstrated that AT2 receptor stimulation activated SHP-1 in fetal VSMCs, whereas AT1 receptor stimulation did not. Transient transfection of a dominant-negative SHP-1 mutant into rat fetal VSMCs resulted in a significant decrease of the AT2 receptor-mediated inhibition of ERK phosphorylation and attenuated the proapoptotic effect of AT2 receptor. CONCLUSION: These results indicate that a crosstalk between AT1 and AT2 receptors regulates the survival of fetal VSMCs and substantiate SHP-1 as a key molecule in AT2 receptor signaling.

Analysis of Variance↗

PubMed2001

ATP-sensitive K+ channels in the hypothalamus are essential for the maintenance of glucose homeostasis.

Glucose-responsive (GR) neurons in the hypothalamus are thought to be critical in glucose homeostasis, but it is not known how they function in this context. Kir6.2 is the pore-forming subunit of K(ATP) channels in many cell types, including pancreatic beta-cells and heart. Here we show the complete absence of both functional ATP-sensitive K+ (K(ATP)) channels and glucose responsiveness in the neurons of the ventromedial hypothalamus (VMH) in Kir6.2-/- mice. Although pancreatic alpha-cells were functional in Kir6.2-/-, the mice exhibited a severe defect in glucagon secretion in response to systemic hypoglycemia. In addition, they showed a complete loss of glucagon secretion, together with reduced food intake in response to neuroglycopenia. Thus, our results demonstrate that KATP channels are important in glucose sensing in VMH GR neurons, and are essential for the maintenance of glucose homeostasis.

ATP-Binding Cassette Transporters↗