Serotonine Bağlı Vasküler Kalsiyum Yanıtlarının Siklopiazonik Asit-Aracılı Potansiyalizasyonuna Protein Kinaz C’nin Katkısı

Ö nceki çalışmalar 5-HT reseptör antagonisti metiserjidin, siklopiazonik asitin CPA potansiyalize ettiği serotonine 5-hidroksitriptamin, 5-HT bağlı kontraksiyonları kısmen inhibe ettiği gösterilmiş ve azalan antagonistik etkiye protein kinaz C PKC -aracılı reseptör internalizasyonunun katkıda bulunduğu önerilmiştir. Bu çalışmada, PKC inhibisyonu varlığında CPA ve 5-HT reseptör antagonistleri metiserjid ve ketanserinin 5-HT’ye bağlı vasküler kalsiyum yanıtları üzerindeki etkileri araştırılmıştır. Bu amaçla A7r5 vasküler düz kas hücrelerindeki hücre içi kalsiyum düzeylerindeki değişimler fluoresan indikatör fura-2 ile spektrofluorometri yöntemi ile belirlenmiştir. Hücreler PKC inhibitörü D-sifingozin ile inkübe edilmiştir. Metodolojik açıdan bakıldığında, aşırı yoğun hücrelerde ajanlara alınan yanıtları gizleyecek düzeyde spontan kalsiyum salınımları belirlenmiştir. 5-HT’ye bağlı kalsiyum yanıtları D-sifingozin ile inkübe edilen hücrelerde kontrol hücrelere göre anlamlı düzeyde azalmıştır. PKC inhibisyonu varlığında CPA, 5-HT’ye bağlı kalsiyum yanıtlarını anlamlı düzeyde potansiyalize etmiştir. Bunun yanı sıra ketanserin, potansiyalize yanıtları kısmen ve anlamlı olmayan düzeyde inhibe etmiştir. Sonuç olarak, hücrelerin aşırı yoğunlukta olması ani kalsiyum tepe spike yanıtlarının oluşmasına neden olabilmekte ve hücre içi kalsiyum düzeylerinin izlendiği çalışmalarda hücrelerin optimum yoğunlukta kullanılmasının önemini göstermektedir. Bulgularımız PKC’nin, 5-HT yanıtları üzerindeki CPA-aracılı potansiyalizasyon ve azalmış antagonistik etkiye kısmen aracılık ettiğini göstermiştir

Anahtar Kelimeler:

CPA, D-sifingozin, kalsiyum, serotonin

The Contribution of Protein Kinase C to Cyclopiazonic Acid- Mediated Potentiation of Serotonin-Induced Vascular Calcium Responses

We previously showed that serotonin 5-Hydroxytryptamine, 5-HT receptor antagonist methysergide partially inhibited cyclopiazonic acid CPA -potentiated and 5-HT-induced contractions suggesting the contribution of protein kinase C PKC -mediated receptor internalization in attenuated antagonism. In the present study, the effects of CPA and 5-HT receptor antagonist methysergide and ketanserin on 5-HT-induced calcium responses following PKC inhibition were further investigated. For this purpose, intracellular calcium levels were monitored in A7r5 vascular smooth muscle cells by spectrofluorometry using the fluorescent indicator fura-2. Cells were pre-incubated with specific PKC inhibitor D-sphingosine. In experimental aspects, spontaneous calcium oscillations hindering the monitoring of responses were determined in over confluent cells. 5-HT-induced calcium levels were significantly decreased in D-sphingosine-treated cells compared to control. CPA significantly potentiated 5-HT-induced calcium responses while ketanserin partially but insignificantly inhibited the potentiated responses in the presence of PKC inhibition. In conclusion, increased cell confluency may result in the generation of spontaneous calcium spikes indicating the importance of using optimum cell density for monitoring of intracellular calcium levels. The data suggests that CPA-mediated potentiation and diminished antagonistic effects on 5-HT-induced calcium elevations in vascular smooth muscle cells, are partially mediated by PKC.

Keywords:

Calcium CPA, D-sphingosine, PKC, Serotonin,

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