Marry Duin

Activation of the phospholipase C pathway by ATP is mediated exclusively through nucleotide type P2‐purinoceptors in C2C12 myotubes

1 The presence of a nucleotide receptor and a discrete ATP‐sensitive receptor on C2C12 myotubes has been shown by electrophysiological experiments. In this study, the ATP‐sensitive receptors of C2C12 myotubes were further characterized by measuring the formation of inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) and internal Ca2+. 2 The nucleotides ATP and UTP caused a concentration‐dependent increase in Ins(1,4,5)P3 content with comparable time courses (EC50: ATP 33 ± 2 μm, UTP 80 ± 4 μm). ADP was less effective in increasing Ins(1,4,5)P3 content of the cells, while selective agonists for P1‐, P2X‐ and P2Y‐purinoceptors, adenosine, α,β‐methylene ATP and 2‐methylthio ATP, appeared to be ineffective. 3 Under Ca2+‐free conditions, the basal level of Ins(1,4,5)P3 was lower than in the presence of Ca2+, and the ATP‐ and UTP‐induced formation of Ins(1,4,5)P3 was diminished. 4 The Ins(1,4,5)P3 formation induced by optimal ATP and UTP concentrations was not additive. ATP‐ and UTP‐induced Ins(1,4,5)P3 formation showed cross‐desensitization, whereas cross‐desensitization was absent in responses elicited by one of the nucleotides and bradykinin. 5 The change in Ins(1,4,5)P3 content induced by effective nucleotides was inhibited by suramin. Schild plots for suramin inhibition of Ins(1,4,5)P3 formation in ATP‐ and UTP‐stimulated myotubes showed slopes greater than unity (1.63 ± 0.09 and 1.37 ± 0.11, respectively). Apparent pA2 values were 4.50 ± 0.48 and 4.41 ± 0.63 for ATP and UTP, respectively. 6 Stimulation of the cells with ATP or UTP induced a rapid increase in intracellular Ca2+, followed by a slow decline to basal levels. Ca2+ responses reached lower maximal values and did not show the slow phase in the absence of extracellular Ca2+. The ATP and UTP‐evoked increase in intracellular Ca2+ was not additive and showed cross‐desensitization. Cross‐desensitization was absent in myotubes stimulated with one of the nucleotides and bradykinin. 7 These results show that ATP‐ and UTP‐induced formation of Ins(1,4,5)P3, Ca2+ release from internal stores and Ca2+‐influx from the extracellular space are mediated exclusively via the nucleotide type P2‐purinoceptor in mouse C2C12 myotubes.

Characterization of P2‐purinoceptor mediated cyclic AMP formation in mouse C2C12 myotubes

1 The formation of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) and inositol(1,4,5)trisphosphate (Ins(1,4,5)P3), induced by ATP and other nucleotides was investigated in mouse C2C12 myotubes. 2 ATP (100 μm) and ATP7S (100 μm) caused a sustained increase in cyclic AMP content of the cells, reaching a maximum after 10 min. The cyclic AMP content reached a maximum in the presence of 100 μm ATP, followed by a decline at higher ATP concentrations. ATP‐induced cyclic AMP formation was inhibited by the P2‐purinoceptor antagonist, suramin. 3 Myotubes hydrolysed ATP to ADP at a rate of 9.7 ± 1.0 nmol mg−1 protein min−1. However, further hydrolysis of ADP to AMP and adenosine was negligible. 4 The cyclic AMP formation induced by ADP (10 μm–1 mm) showed similar characteristics to that induced by ATP, but a less pronounced decline was observed than with ATP. ADP‐induced cyclic AMP formation was blocked by suramin, while cyclic AMP formation elicited by adenosine (10 μm–1 mm) was insensitive to suramin. 5 The ATP analogue, α,β‐methylene‐ATP also induced a suramin‐sensitive cyclic AMP formation, while 2‐methylthio‐ATP and the pyrimidine, UTP, did not affect cyclic AMP levels. 6 Stimulation of the myotubes with ATP or UTP (10 μm–1 mm) caused a concentration‐dependent increase in the Ins(1,4,5)P3 content of the cells. ADP (100 μm–1 mm) was less effective. Adenosine did not affect Ins(1,4,5)P3 levels. 7 Incubation of the cells with UTP (30 μm–1 mm) inhibited the ATP‐ and ADP‐induced cyclic AMP formation, suggesting that stimulation of the ‘nucleotide’ type P2‐receptor inhibits P2‐purinoceptor mediated cyclic AMP formation in C2C12 myotubes. In contrast, UTP (30 μm–1 mm) enhanced adenosine‐induced cyclic AMP formation. 8 Adenosine‐sensitive P1‐purinoceptors activating cyclic AMP formation were found in C2C12 myotubes. Further, a novel P2‐purinoceptor is postulated, sensitive to ATP, ADP and ATPγS, which also activates the formation of cyclic AMP in C2C12 myotubes.

α1-Adrenoceptor-mediated contraction of rat aorta is partly mediated via transactivation of the epidermal growth factor receptor

Background and purpose:  High level of plasma catecholamines is a risk factor for vascular diseases such as hypertension and atherosclerosis. Catecholamines induce hypertrophy of vascular smooth muscle through α1‐adrenoceptors, which in cell culture involves the transactivation of epidermal growth factor receptor (EGFR). We hypothesized that EGFR transactivation was also involved in contractions of rat aorta mediated by α1‐adrenoceptors.

Regulation of histamine‐ and UTP‐induced increases in Ins(1,4,5)P3, Ins (1,3,4,5)P4 and Ca2+ by cyclic AMP in DDT1 MF‐2 cells

1 Stimulation of P2U‐purinoceptors with UTP or histamine H1‐receptors with histamine gave rise to the formation of inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) and inositol 1,3,4,5‐tetrakisphosphate (Ins(1,3,4,5)P4) in DDT1 MF‐2 smooth muscle cells. 2 Stimulation of P2U‐purinoceptors or histamine H1‐receptors caused an increase in cytoplasmic Ca2+, consisting of an initial peak, representing the release of Ca2+ from internal stores and a sustained phase representing Ca2+ influx. 3 The P2U‐purinoceptor‐mediated Ca2+‐entry mechanism was more sensitive to UTP than Ca2+‐mobilization (EC50: 3.3 μm ± 0.4 μm vs 55.1 μm ± 9.2 μm), in contrast to these processes activated by histamine H1‐receptors (EC50: 5.8 μm ± 0.6 μm VS 3.1 μm ± 0.5 μm). 4 Pre‐stimulation of cells with several adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) elevating agents, reduced the histamine H1‐receptor‐mediated formation of Ins(1,4,5)P3 and Ins(1,3,4,5)P4. Forskolin completely inhibited Ins(1,4,5)P3 formation (IC50: 158 ± 24 nm) whereas Ins(1,3,4,5)P4 formation was inhibited by only 45% (IC50: 173 ± 16 nm). The P2U‐purinoceptor‐mediated production of these inositol phosphates was not affected by cyclic AMP. 5 Forskolin and isoprenaline reduced the histamine‐induced increase in cytoplasmic Ca2+, as measured in Ca2+ containing medium and in nominally Ca2+‐free medium but did not change the UTP‐induced increase in cytoplasmic Ca2+. 6 These results clearly demonstrate that cyclic AMP differentially regulates components of the histamine induced phospholipase C signal transduction pathway. Furthermore, cyclic AMP does not affect the phospholipase C pathway activated by stimulation of P2U‐purinoceptors in DDT1 MF‐2 cells.

CALCIUM RELEASE FROM SEPARATE RECEPTOR-SPECIFIC INTRACELLULAR STORES INDUCED BY HISTAMINE AND ATP IN A HAMSTER-CELL LINE

1. The specificity of intracellular Ca2+ stores to Ca(2+)‐mobilizing agonists was studied in DDT1 MF‐2 vas deferens cells of the Syrian hamster. 2. Application of histamine (100 microM) or ATP (100 microM) to the DDT1 MF‐2 cells caused an initial increase of intracellular Ca2+ followed by a lower phase as measured by using Indo‐1 as fluorescent probe at 22 degrees C. The basal Ca2+ level (146 nM) was enhanced to 309 nM by histamine and to 379 nM by ATP. 3. A transient rise in intracellular Ca2+ lasting for about 2 min was measured in the presence of histamine or ATP in the absence of extracellular Ca2+. The basal Ca2+ level (78 nM) was increased to 128 nM by histamine and to 145 nM by ATP. 4. A transient hyperpolarization was elicited in single cells as measured with microelectrodes by both agonists under Ca(2+)‐free conditions with a similar time course as the change in internal Ca2+. The hyperpolarization observed in the presence of histamine amounted to 23 mV and 31 mV with ATP. The histamine‐induced responses were abolished by the H1 histaminoceptor antagonist mepyramine (10 microM) and the responses evoked by ATP were blocked by the P2 purinoceptor antagonist suramin (300 microM). 5. A second internal Ca2+ response could only be evoked under Ca(2+)‐free conditions by applying a higher agonist concentration or after replenishing the intracellular stores with Ca2+ from the extracellular space. 6. A second addition of an optimal concentration (100 microM) of the agonist to the cells under Ca(2+)‐free conditions did not evoke mobilization of internal Ca2+ or hyperpolarization, but resulted in a rise of the cellular inositol (1,4,5)‐trisphosphate content (Ins(1,4,5)P3) as determined by a radioligand binding assay. 7. The cells responded to both agonists (100 microM) with a transient Ca2+ response if successively applied at a maximal effective concentration (100 microM) under Ca(2+)‐free conditions. 8. Simultaneous stimulation of H1 histaminoceptors and P2 purinoceptors resulted in the absence of external Ca2+ in an additional increase in internal Ca2+ represented by the amplitude and area of the response and in an increased response area of the hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

P2 –Purinoceptor-mediated inositol phosphate formation in relation to cytoplasmic calcium in DDT1 MF-2 smooth muscle cells

The effect of P2 purinoceptor stimulation on inositol phosphate (InsP) formation in relation to the intracellular Ca2+ concentration was measured in vas deferens DDT1 MF-2 smooth muscle cells. The different [3H]myo-inositol-labelled InsP fractions were analyzed by high performance liquid chromatography and intracellular Ca2+ was determined by measuring fluorescence using Indo-1 as indicator. Stimulation with ATP (10(-4) M) resulted in an enhanced formation of inositol mono-, bis-, tris- and tetrakisphosphate (InsP1, InsP2, InsP3 and InsP4), but no changes occurred in the formation of inositol pentakis- and hexakisphosphate (InsP5 and InsP6). The putative second messenger Ins(1,3,4,5)P4 rapidly increased after addition of the agonist, reaching a maximum after about 2 min. The isomer Ins(1,4,5)P3 showed a delayed rise starting after about 2 min. The formation of Ins(1,3,4,5)P4 in the presence of ATP (2 min) was concentration-dependent, reaching a half maximal value at about 50 microM of the agonist. The intracellular Ca2+ concentration showed an initial increase after P2 purinoceptor stimulation, reaching a plateau after 2 min. Both the top of the initial phase and the plateau value of the response reached a half maximal value at an ATP concentration of about 7 microM. This Ca2+ response could be evoked repeatedly by ATP and was not affected by diltiazem (10(-5) M). In the absence of external Ca2+, the internal Ca2+ concentration increased transiently in the presence of ATP without showing the plateau phase. This response could be evoked only once under Ca2(+)-free conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidermal Growth Factor Receptor Inhibitor PKI-166 Governs Cardiovascular Protection without Beneficial Effects on the Kidney in Hypertensive 5/6 Nephrectomized Rats

Transactivation of epidermal growth factor receptor (EGFR) signaling by G protein–coupled receptors has been implicated in several cardiovascular (CV) conditions, including hypertension, heart failure, and cardiac and vascular hypertrophy. However, the therapeutic potential of EGFR inhibition in these conditions is currently unknown. The main objective of the present study was to investigate cardiac, vascular, and renal effects of EGFR inhibition by 4-[4-[[(1R)-1-phenylethyl]amino]-7H-pyrrolo[2,3-d]pyrimidin-6-yl]phenol (PKI-166) in the hypertensive chronic kidney disease model. Rats underwent 5/6 nephrectomy (5/6Nx) and were treated with PKI-166, lisinopril or vehicle from week 6 after disease induction until week 12. Sham animals received either PKI-166 or vehicle. Treatment with PKI-166 did not affect the development of the characteristic renal features in 5/6Nx, including proteinuria, diminished creatinine clearance, and increased glomerulosclerosis, whereas these were attenuated by lisinopril. Despite absence of effects on progressive renal damage, PKI-166 attenuated the progression of hypertension and maintained cardiac function (left ventricle end-diastolic pressure) to a similar extent as lisinopril. Also, PKI-166 attenuated the increase in phosphorylated EGFR in the heart as induced by 5/6Nx. Moreover, PKI-166 and lisinopril restored the impaired contraction of isolated thoracic aortic rings to phenylephrine and angiotensin II and impaired myogenic constriction of small mesenteric arteries in 5/6Nx rats. Blockade of the EGFR displays a CV benefit independent of limiting the progression of renal injury. Our findings extend the evidence on EGFR signaling as a target in CV disorders.

Genetic deletion of growth differentiation factor 15 augments renal damage in both type 1 and type 2 models of diabetes

Growth differentiation factor 15 (GDF15) is emerging as valuable biomarker in cardiovascular disease and diabetic kidney disease. Also, GDF15 represents an early response gene induced after tissue injury and studies performed in GDF15 knockout (KO) mice suggest that GDF15 plays a protective role after injury. In the current study, we investigated the role of GDF15 in the development of diabetic kidney damage in type 1 and type 2 models of diabetes. Renal damage was assessed in GDF15 KO mice and wild-type (WT) mice in streptozotocin type 1 and db/db type 2 diabetic models. Genetic deletion of GDF15 augmented tubular and interstitial damage in both models of diabetes, despite similar diabetic states in KO and WT mice. Increased tubular damage in KO animals was associated with increased glucosuria and polyuria in both type 1 and type 2 models of diabetes. In both models of diabetes, KO mice showed increased interstitial damage as indicated by increased α-smooth muscle actin staining and collagen type 1 expression. In contrast, glomerular damage was similarly elevated in diabetic KO and WT mice. In type 1 diabetes, GDF15 KO mice demonstrated increased expression of inflammatory markers. In type 2 diabetes, elevated levels of plasma creatinine indicated impaired kidney function in KO mice. GDF15 protects the renal interstitium and tubular compartment in experimental type 1 and 2 diabetes without affecting glomerular damage.

Different mechanisms of Ca2(+)-handling following nicotinic acetylcholine receptor stimulation, P2U-purinoceptor stimulation and K(+)-induced depolarization in C2C12 myotubes.

1 The increase in intracellular Ca2+ on nicotinic acetylcholine receptor (nAChR) stimulation, P2U‐purinoceptor stimulation and K+‐induced depolarization was investigated in mouse C2C12 myotubes by use of fura‐2 fluorescence to characterize the intracellular organisation of Ca2+ releasing stores and Ca2+‐entry process. 2 Stimulation of nAChRs with carbachol induced a rapid rise in internal Ca2+ (EC50 = 0.85±0.09 μm), followed by a sustained phase. The Ca2+ response evoked by carbachol (10 μm) was completely blocked by the nAChR antagonist, pancuronium (3 μm), but was not affected by the muscarinic antagonist, atropine (3 μm), or under conditions when Ca2+ entry was blocked by La3+ (50 μm) or diltiazem (10 μm). Addition of pancuronium (3 μm) during the sustained phase of the carbachol‐evoked response did not affect this phase. 3 Stimulation of P2U purinoceptors with ATP (1 mM) induced a somewhat higher biphasic Ca2+ response (EC50 of the rapid phase: 8.72±0.08 μm) than with carbachol. Pretreatment with La3+ abolished the sustained phase of the ATP‐induced Ca2+ response, while the response was unaffected by diltiazem or pancuronium. 4 Stimulation of the cells with high K+ (60 mM), producing the same depolarization as with carbachol (10 μm), induced a rapid monophasic Ca2+ response, insensitive to diltiazem, pancuronium or La3+. 5 Under Ca2+‐free conditions, the sustained phase of the carbachol‐ and ATP‐evoked responses were abolished. Pre‐emptying of depolarization‐sensitive stores by high K+ under Ca2+‐free conditions did not affect the carbachol‐ or ATP‐evoked Ca2+ mobilization and vice versa. Preincubation of the cells with ATP in the absence of extracellular Ca2+ decreased the amplitude of the subsequent carbachol‐induced Ca2+ response to 11%, while in the reverse procedure the ATP‐induced response was decreased to 65%. Ca2+ mobilization evoked by simultaneous addition of optimal concentrations of carbachol and ATP was increased compared to levels obtained with either agonist. 6 Preincubation with high K+ under normal conditions abolished the sustained phase of the ATP‐evoked Ca2+ response. The carbachol response consisted only of the sustained phase in the presence of high K+. 7 The carbachol‐induced Ca2+ response was completely abolished under low Na+/Ca2+‐free conditions, while under low Na+ conditions only a sustained Ca2+ response was observed. The ATP‐ and K+‐induced responses were changed compared to Ca2+‐free conditions. 8 ATP (300 μm) induced the formation of Ins(1,4,5)P3 under Ca2+‐free conditions with a comparable time course to that found for the rise in internal Ca2+. In contrast to ATP, carbachol (10 μm) did not affect Ins(1,4,5)P3 levels under Ca2+‐free conditions. 9 It is concluded that the Ca2+ release from discrete stores of C2C12 myotubes is induced by stimulation of nAChRs, P2U‐purinoceptors and by high K+. Only the P2U‐purinoceptor and nAChR activated stores show considerable overlap in releasable Ca2+. Sustained Ca2+‐entry is activated by stimulation of nAChRs and P2U‐purinoceptors via separate ion‐channels, which are different from the skeletal muscle nAChR‐coupled cation‐channel.

Intracellular Transactivation of Epidermal Growth Factor Receptor by α1A-Adrenoceptor Is Mediated by Phosphatidylinositol 3-Kinase Independently of Activation of Extracellular Signal Regulated Kinases 1/2 and Serine-Threonine Kinases in Chinese Hamster Ovary Cells

Transactivation of epidermal growth factor receptor (EGFR) by α1-adrenoceptor (α1-AR) is implicated in contraction and hypertrophy of vascular smooth muscle (VSM). We examine whether all α1-AR subtypes transactivate EGFR and explore the mechanism of transactivation. Chinese hamster ovary (CHO) cells stably expressing one subtype of α1-AR were transiently transfected with EGFR. The transactivation mechanism was examined both by coexpression of a chimeric erythropoietin (EPO)-EGFR with an extracellular EPO and intracellular EGFR domain, and by pharmacologic inhibition of external and internal signaling routes. All three α1-AR subtypes transactivated EGFR, which was dependent on the increase in intracellular calcium. The EGFR kinase inhibitor AG1478 [4-(3′-chloroanilino)-6,7-dimethoxyquinazoline] abrogated α1A-AR and α1D-AR induced phosphorylation of EGFR, but both the inhibition of matrix metalloproteinases by GM6001 [(R)-N4-hydroxy-N1-[(S)-2-(1H-indol-3-yl)-1-methylcarbamoyl-ethyl]-2-isobutyl-succinamide] or blockade of EGFR by cetuximab did not. Stimulation of α1A-AR and α1D-AR also induced phosphorylation of EPO-EGFR chimeric receptors. Moreover, α1A-AR stimulation enhanced phosphorylation of extracellular signal regulated kinase (ERK) 1/2 and serine-threonine kinases (Akt), which were both unaffected by AG1478, indicating that ERK1/2 and Akt phosphorylation is independent of EGFR transactivation. Accordingly, inhibitors of ERK1/2 or Akt did not influence the α1A-AR–mediated EGFR transactivation. Inhibition of calcium/calmodulin-dependent kinase II (CaMKII), phosphatidylinositol 3-kinase (PI3K), and Src, however, did block EGFR transactivation by α1A-AR and α1D-AR. These findings demonstrate that all α1-AR subtypes transactivate EGFR, which is dependent on an intracellular signaling route involving an increase in calcium and activation of CaMKII, PI3K, and Src, but not the of ERK1/2 and Akt pathways.